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Annals of the ICRP

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ICRP PUBLICATION 1XX

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Stem Cell Biology with Respect toCarcinogenesis Aspects of RadiologicalProtection
Editor-in-Chief
C.H. CLEMENT
Associate Editor
N. HAMADA

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Authors on behalf of ICRP
O. Niwa, M.H. Barcellos-Hoff, R.K. Globus, J.D. Harrison,
J.H. Hendry, P. Jacob, M.T. Martin, T.M. Seed, J.W. Shay,
M.D. Story, K. Suzuki, S. Yamashita

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PUBLISHED FOR

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  The International Commission on Radiological Protection

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by

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[SAGE logo]

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Please cite this issue as ‘ICRP, 201X. Stem Cell Biology with Respect toCarcinogenesis Aspects of Radioligical Protection. ICRP Publication 1XX, Ann.
ICRP 4X(0).’

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CONTENTS	  ABSTRACT.............................................................................................................................. 4
PREFACE ................................................................................................................................. 6
EXECUTIVE SUMMARY ...................................................................................................... 8
GLOSSARY ........................................................................................................................... 12
ABBREVIATIONS ................................................................................................................ 23
1. INTRODUCTION ............................................................................................................. 25
1.1. Purpose of the report ................................................................................................... 25
1.2. Radiation carcinogenesis models and stem cell biology............................................. 26
2. GENERAL FEATURES OF TISSUE STEM CELLS ...................................................... 28
2.1. Cell division and differentiation in adult tissues......................................................... 28
2.2. Functional identification and isolation of tissue stem cells ........................................ 31
2.3. Radiosensitivity and DNA damage response of tissue stem cells .............................. 33
2.4. Ageing and exhaustion of tissue stem cells ................................................................ 39
2.5. Tissue stem cell niche ................................................................................................. 41
3. THE ROLE OF TISSUE STEM CELLS IN RADIATION CARCINOGENESIS ........... 46
3.1. The role of stem cells in radiation carcinogenesis ...................................................... 46
3.2. Stem cells and stem cell niche in radiation carcinogenesis ........................................ 53
3.3. Dose rate effect for radiation carcinogenesis .............................................................. 55
3.4. Experimental animal studies to supplement human data ............................................ 57
3.5. Location of target cells in a tissue............................................................................... 61
3.6. Cell-based and tissue-based considerations ................................................................ 62
3.7. Age dependence of radiation carcinogenesis .............................................................. 64
3.8. Summary ..................................................................................................................... 69
3.9. Recommendations for future research ........................................................................ 71
ANNEX A: HAEMATOPOIETIC TISSUES: ROLE PLAYED BY STEM CELLS AND
LINEAGE-COMMITTED PROGENITOR CELLS IN RADIATION-INDUCEDLEUKAEMIA ............................................................................................................. 72
A.1. Radiation-induced leukaemias and lymphomas ......................................................... 72
A.2. Relevant data for various radiations and exposure types ........................................... 77
A.3. General features of haematopoietic tissues ................................................................ 78
A.4. Radiosensitivity.......................................................................................................... 87
A.5. Characteristics of single-cell responses ..................................................................... 91
A.6. Mutagenesis ............................................................................................................... 92
A.7. Summary and conclusions ......................................................................................... 98
ANNEX B: MAMMARY GLAND STEM CELLS ............................................................ 101
B.1. Radiation-induced breast cancer .............................................................................. 101
B.2. General features of the mammary gland and carcinogenesis ................................... 104
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B.3. Radiosensitivity ........................................................................................................ 114
B.4. Experimental models of carcinogenesis ................................................................... 116
B.5. Summary and conclusions........................................................................................ 120
ANNEX C: THYROID STEM CELLS ............................................................................... 122
C.1. Radiation-induced thyroid carcinogenesis ............................................................... 122
C.2. Human data on radiation qualities and type of exposure ......................................... 123
C.3. General features of the thyroid ................................................................................. 125
C.4. Radiosensitivity ........................................................................................................ 130
C.5. Mutagenesis.............................................................................................................. 131
C.6. Summary .................................................................................................................. 134
ANNEX D: DIGESTIVE TRACT STEM CELLS.............................................................. 136
D.1. Radiation induced cancer in the digestive tract ....................................................... 136
D.2. General features of the digestive tract...................................................................... 138
D.3. Radiosensitivity........................................................................................................ 147
D.4. Mutagenesis ............................................................................................................. 152
D.5. Target cells and radiation protection ........................................................................ 156
D.6. Summary .................................................................................................................. 158
ANNEX E. LUNG STEM CELLS ...................................................................................... 159
E.1. Lung cancer .............................................................................................................. 159
E.2. Radiation induction of lung cancer in humans ......................................................... 160
E.3. General architectural features of lung tissue ............................................................ 164
E.4. Radiosensitivity: Cells involved in lung repair after damage, and radiation as a lungcarcinogen ................................................................................................................. 178
E.5. Models of carcinogenesis bridging the stem cell concept and epidemiology .......... 181
ANNEX F: SKIN STEM CELLS AND RADIATION CARCINOGENESIS.................... 182
F.1. Skin carcinogenesis .................................................................................................. 182
F.2. Radiation carcinogenesis in human skin................................................................... 184
F.3. Radiation carcinogenesis in rodent skin ................................................................... 188
F.4. General features of skin ............................................................................................ 191
F.5. Cellular origin of skin tumours ................................................................................. 200
F.6. Radiosensitivity of stem cells and progenitors ......................................................... 204
F.7. Mutagenesis .............................................................................................................. 209
F.8. Summary and conclusions ........................................................................................ 209
ANNEX G: BONE STEM CELLS ...................................................................................... 211
G.1. Radiation carcinogenesis.......................................................................................... 211
G.2. General features of bone .......................................................................................... 214
G.3. Radiosensitivity........................................................................................................ 222
G.4. Mutagenesis ............................................................................................................. 224
G.5. Summary .................................................................................................................. 224
REFERENCES ..................................................................................................................... 226

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ABSTRACT

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Stem Cell Biology with Respect to Carcinogenesis Aspectsof Radiological Protection

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ICRP Publication 1XX

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Approved by the Commission in Month 201Y

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Abstract- This report provides a review of stem cells/progenitor cells and their responses toionising radiation in relation to issues relevant to stochastic effects of radiation which form amajor part of the ICRP system of radiation protection. Current information on stem cellcharacteristics, maintenance and renewal, evolution with age, location in stem cell “niches”,
radiosensitivity to acute and protracted exposures, is presented in a series of substantial
reviews as Annexes concerning haematopoietic tissue, mammary gland, thyroid, digestive
tract, lung, skin and bone. This foundation of knowledge of stem cells is used in the main textof the report to provide a biological basis to issues such as the linear-no-threshold (LNT)
model, cancer risk among tissues, dose-rate effects and changes in the risk of radiation
carcinogenesis by age at exposure and attained age.
Knowledge of the biology and associated radiation biology of stem cells and progenitor
cells is more developed in tissues which renew fairly rapidly, such as haematopoietic tissue,
intestinal mucosa, and epidermis, although all the tissues considered here possess stem cell
populations. Important features of stem cell maintenance, renewal and response are themicroenvironmental signals operating in the niche residence which also has a well-defined
spatial location identified in some tissues. The identity of the target cell for carcinogenesis
continues to point to the more-primitive stem cell population which is mostly quiescent andhence able to accumulate the protracted sequence of mutations necessary to result inmalignancy. There is some potential for daughter progenitor cells also to be target cells inparticular cases, e.g. in haematopoietic tissue. Several biological processes could contributein protecting stem cells from mutation accumulation: rapid induced death of injured stem
cells, and retention of the DNA parental template strand during divisions in some tissue
systems, so that mutations are passed to the daughter differentiating cells and not retained in
the parental cell. Also, stem cell competition, whereby undamaged stem cells outcompete
damaged stem cells for residence in the niche during long protracted irradiations.
The aforementioned processes may contribute to the carcinogenic radiation risk values
among tissues, and may help explain why a rapidly-replicating tissue such as small intestineis free of such risk. They may also provide mechanistic understanding which relates to thedose and dose-rate effectiveness factor (DDREF) currently used in radiation protection
guidelines. It is noteworthy that DNA repair operates mainly within a few days after
irradiation while stem cell competition is a slow process requiring weeks and/or many
months depending on the tissue type. These distinctions are likely to be of importance when
considering the differences in the conclusions of human epidemiological studies of chronic
radiation scenarios, where some organs and tissues demonstrate no risk up to and above a few

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hundred mSv, while others demonstrate a risk at moderate doses comparable to those for
acute radiation exposures.
© 201Y ICRP. Published by SAGE.

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AUTHORS ON BEHALF OF ICRPO. NIWA, M.H. BARCELLOS-HOFF, R.K. GLOBUS, J.D. HARRISON,
J.H. HENDRY, P. JACOB, M.T. MARTIN, T.M. SEED, J.W. SHAY,
M.D. STORY, K. SUZUKI, S. YAMASHITA

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Keywords: Stem cells; Normal tissues; Radiation sensitivity; Chronic radiation;
Carcinogenesis; Radiation risk

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PREFACEThe present report was prepared by a task group of ICRP Committee 1, under thefollowing terms of reference: (a) to review stem cell biology and radiobiology with reference
to mechanisms of radiation carcinogenesis; (b) to compare the response of stem andassociated cells in different tissues with the respective risks of cancer, and elucidate the likely
roles of stem cells, progenitor cells, and the stem-cell niche; and (c) to use knowledge ofstem-cell/progenitor-cell biology, radiation responses and carcinogenic risks fromhomogeneous acute exposures, in a discussion of the guiding principles for the projection ofstochastic risks for short-range-radiation and chronic-irradiation scenarios.
In its recommendations and various reports, the ICRP has made some judgements andassumptions about the location and radiation response of the target cells responsible for
carcinogenesis in various tissues. In most cases the target cells are considered to be the tissue
stem cells, and in some cases also their daughter progenitor cells. The renewal and radiation
response of these cells change with age and are governed by signals from their “niche”
residence. The fundamental evidence for stem cells as target cells has been increasing inrecent years. This evidence contributes to the understanding of the biological basis for
carcinogensis, and helps to support modelling of human responses. It was considered that areport on this subject of target/stem cells would be topical and valuable in order to put all thetarget cell evidence for carcinogenic radiation risk in different tissues into a common
framework and perspective for the first time.
In order to address stem cell knowledge with respect to particular issues of continuing
importance to the ICRP, such as the linear-no-threshold (LNT) model, dose rate effects,
location of target cells, tissue risk factors, and age dependent sensitivity to radiation, it wasnecessary to review evidence for different organ systems. This was undertaken by compilinga series of Annexes as separate reviews using a common template of topics, for each of achosen series of seven organ systems with different characteristics. The Annexes (andauthors) comprise: Annex A. Haematopoietic tissues: role played by stem cells and lineagecommitted progenitors in radiation-induced leukaemia (T.M. Seed); Annex B. Mammary
gland stem cells (M.H. Barcellos-Hoff); Annex C. Thyroid stem cells (K. Suzuki and S.
Yamashita); Annex D. Digestive tract stem cells (J.H. Hendry); Annex E. Lung stem cells(J.W. Shay, M.D. Story, and P. Jacob); Annex F: Skin stem cells and radiation carcinogenesis(M.T. Martin); and Annex G: Bone stem cells (J.D. Harrison and R.K. Globus). Information
on both humans and experimental animal systems was reviewed, and projections were madeof the possible role of various stem cell processes in cancer risk.
The membership of the Task Group was as follows:
O. Niwa (Chair)
M.H. Barcellos-Hoff

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M.T. Martin
K. Suzuki

The corresponding members were:
J.D. Harrison
S. Yamashita

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J.H. Hendry
T.M. Seed

J.W. Shay
M.D. Story

P. Jacob
R.K. Globus

Main Commission critical reviewers were:

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J.D. Boice Jr
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Jai-Ki Lee

Many helpful comments were received in particular from R. Cox and C. S. Potten(deceased 3 August 2012), Committee 1 members S. Bouffler, D. Laurier, A.J. Sigurdson, M.
Tirmarche, R. Wakeford, W. Doerr, as well as C. Land, N. Nakamura, A. Noda, D. Preston, J.
Preston, and R. Shore.
The membership of Committee 1 during the period of preparation of this report was:(2009-2013)
J. Preston (Chair)
T.V. Azizova
R. Chakraborty
S.C. Darby
F.A. Stewart

W.F. Morgan (Vice-Chair)
N. Nakamura
W. Rühm
S. Salomaa
A.J. Sigurdson

J.H. Hendry
D.O. Stram
M. Tirmarche
R. Wakeford
P.-K. Zhou

(2013-2017)
W.F. Morgan (Chair)
T.V. Azizova
N. BanS. Bouffler
R. Chakraborty
W. Doerr

A.J. Sigurdson (Vice-Chair)
M. Hauptmann
D. Laurier
P. Rajaraman
S. Salomaa
D.O. Stram

W. Rühm
Q. SunM. Tirmarche
R. Wakeford

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EXECUTIVE SUMMARY(a) The Commission issued new recommendations for a System of Radiological Protectionin Publication 103 (ICRP, 2007). Stochastic effects of radiation, which are cancer andheritable effects, were also considered. Previously, the Commission reviewed various aspectsof cancer induction from radiation, for example Skin Cancer Risk in Publication 59 (ICRP,
1992), Genetic Susceptibility to Cancer in Publication 79 (ICRP, 1998), and Low-dose
Extrapolation of Radiation-related Cancer Risk in Publication 99 (ICRP, 2005). Morerecently the Commission also reviewed Lung Cancer Risk from Radon in Publication 115(ICRP, 2010) and the Threshold Doses for Tissue Reactions (Deterministic effects) in
Publication 118 (ICRP, 2012).
(b) Cancers arise as a result of mutational changes in single target cells, and an aspect offundamental importance is the nature and characteristics of the target cells responsible forcancer induction. Previously it has been assumed that in most cases the target cells are the“stem cells” in the tissue in question, without further elaboration. In recent years there has
been an enormous increase in knowledge about the lineages of stem cells, daughterprogenitor cells and the differentiated functional cells. Knowledge has accumulated also
about the regulatory signals in different tissues and how the tissues respond to injury. Hence
it was considered appropriate at this time to review those aspects of the field of stem cells andtheir regulatory processes, which are pertinent to radiation carcinogenesis models. However,there are no recent comprehensive reviews of carcinogenic mechanisms after exposure toionising radiation, regarding stem cells as target cells in different tissues and including age
effects.
(c) The main text of the report consists of Chapters 1-3. These are followed by Annexes AG which consist of detailed reviews on stem cells in the haematopoietic system, mammary
gland, thyroid, digestive tract, lung, skin and bone. These particular tissues were chosen on
the basis of differing tissue risk coefficients and weighting factors (wT), and the fact that
information on the stem cell systems for haematopoiesis, intestine, and skin is well developed,
and less so for mammary gland, thyroid, lung and bone.
(d) Chapter 1 describes the objectives of the report and lists main questions in relation toradiation carcinogenesis models and stem cell biology: What are the target cells for
carcinogenesis, and where are they located? Does the LNT model fit with considerations ofstem-cell-based radiation carcinogenesis, and if so, how? Is the current DDREF value
supported by information concerning stem-cell-based radiation carcinogenesis? What are themechanisms related to the stem cell response, and do these mechanisms help to explain thetissue differences in the sensitivity to radiation carcinogenesis? What could be an underlying
mechanism related to stem cells for the age-dependent sensitivity to radiation carcinogenesis,and hence radiation risk?
(e) Chapter 2 gives the general features of tissue stem cells, including cell division anddifferentiation in adult tissues, functional identification and isolation of stem cells, theirradiosensitivity and DNA damage response, ageing and exhaustion aspects of tissue stem
cells, and the very important stem-cell “niche” residence where they are maintained andregulated (see Fig. 2.1.). It has become clear that in the tissues with long lineages, there is ahierarchical spectrum of stem-cell stages with differing characteristics of quiescence(dormancy), renewal, radiosensitivity, propensity to apoptosis, and replication-mediatedmutation rates.
(f) Two biological mechanisms specific to stem cells have been described which may play arole in cancer incidences from protracted compared to acute exposures. The first is the8DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
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immortal-DNA-strand hypothesis, whereby the parental DNA template is retained duringstem-cell divisions so that their mutational load is kept low, and this would act to protect
against carcinogenesis. There is evidence in support of this mechanism in small intestinal
crypts, mammary epithelium, some muscle satellite cells and progenitor cells, and some
central nervous system (CNS) cells. Supportive evidence was also inferred from studies of
tongue epithelium, and it could be the mechanism behind DNA-label retention in epidermis
but probably not the hair follicle bulge region. However, the mechanism has been found not
to apply in haematopoietic stem cells (HSCs). Hence although there is evidence for it invarious tissues, it apparently does not apply universally. Another important feature describedrecently is the concept of competition between normal and radiation-injured stem cells forresidence in the niche. New studies suggest that the niche, i.e. the signal-originating
microenvironment, which controls stem cell behaviour, may be susceptible to damage. Niche
integrity provides another biological mechanism for keeping low the mutation load fromprotracted and chronic radiation exposures, which again is tissue-dependent.
(g) Chapter 3 discusses the role of tissue stem cells in radiation carcinogenesis, and those
aspects which are pertinent to key issues like the LNT model, DDREF, and age-related
effects. Concerning LNT, the dose responses for incidence and mortality from all solidcancers among atomic bomb survivors in general do not show a significant deviation fromlinearity (notable exceptions are skin and bone, and several other organs and tissues which do
not show significant increases in radiation risk such as the testes, pancreas and uterine cervix).
Some animal data tend to support the LNT model, although again there are several exceptions.
The features of dose-dependent non-targeted and epigenetic effects, and adaptation,
demonstrated in various cellular systems, may play a role in the radiation responses, but it is
not yet clear how to incorporate these phenomena into a protection framework.
(h) The excess risk of cancer relates to carcinogenesis of target cells. Carcinogenesis
depends primarily on three mechanistic factors: (1) the number and sensitivity of target cells
to radiation induced mutation; (2) the retention of mutated target cells in a tissue; and (3) the
population size of target cells with a sufficient number of predisposing mutations. The excess
absolute risk (EAR, the additional risk above the baseline) function is obtained by subtracting
the baseline risk of an unexposed population from the overall risk of the exposed population,and therefore it reflects the radiation-induced cancers in an exposed population which are
directly related to the number of predisposed target cells. On the other hand, the excess
relative risk (ERR, the proportional increase in risk above baseline) function is alreadynormalised for the background risk and therefore reflects the sensitivity of a tissue toradiation carcinogenesis for which the sensitivity of target cells to mutagenesis and theirretention in the tissue are contributing factors.
(i) The numerical value(s) of DDREF has been under much discussion recently.
Calculation-based evaluation of DDREF uses a linear-quadratic (LQ) model which assumes asimilarity of the low dose effect and the low dose rate effect. However, from the information
reviewed in the present report, it is clear that the factors DEF (dose effectiveness factor) andDREF (dose rate effectiveness factor) are conceptually different at the biological level. The
former applies for low acute doses, and the latter applies for low protracted doses where
additional exposure-duration processes may modify target/stem cell responses, e.g. DNA
repair kinetics, template-DNA retention, and stem-cell/niche competition processes. It isnoteworthy that DNA repair operates mainly within a few days after irradiation while stem
cell competition is rather a slow process requiring weeks and/or months depending on thetissue type. In this respect, the two mechanisms operate at different dose rates. Also, lowchronic radiation exposure of humans is often concomitant with, or subsequent to, exposure
to other carcinogenic agents, which may also affect stem cell systems. This may influence the9DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
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background rate of cancer in a given population and affect not only projections of doseresponse relationships from acute to chronic exposure scenarios but also comparisons ofexperimental animal data with human epidemiological studies. These distinctions suggest that
more emphasis should be given to epidemiological data, which arise from chronic radiation
scenarios, rather than relying on risk projections to lower protracted doses from acute higherdose scenarios.
(j) The concept of stem cell competition is useful in understanding the behaviour ofradiation exposed stem cells. Interestingly, lymphocytes from in utero exposed atomic bomb
survivors and in-utero-exposed mice largely lacked chromosome aberrations after moderate
doses of radiation, suggesting a possible competition-mediated elimination of aberrant
haematopoietic stem cells. In contrast, stem cell competition is likely to be less stringent
during childhood growth, when the stem-cell/niche units increase in number to cope with theincrease in the tissue volume during childhood growth. Such behaviour of irradiated stem
cells might have relevance to the age-dependent sensitivity to radiation carcinogenesis.
(k) In the Annexes, the role of stem cells is discussed in detail for a number of tissues.
Annex A describes the extensive knowledge of the three haematopoietic cell hierarchies(erythroid, granuloid, and lymphoid), and their dependent stem cells. In mammary glandepithelium there is much evidence for the presence of a very small stem cell population(Annex B), and various markers are now available for those cells. The sensitivity to radiation
carcinogenesis may be influenced by the irradiated microenvironment, in which specificsignals are induced that affect stem cell regulation. In thyroid epithelium (Annex C) there isevidence for the presence of a stem-cell-type lineage, but the niche location and signalling
pattern in the thyroid follicles are not yet described. The target cells for colonic tumours(Annex D) are considered to be the crypt stem cells, and the potential inclusion of daughterprogenitor cells as target cells for radiation is not yet resolved. Genetically-controlled
differences in susceptibility to stem-cell death in small versus large intestine may play a rolein the lack of radiation-induced tumours in the small intestine. In the lung (Annex E),
multipotent cells exist in the bronchiolar-alveolar duct junction zone (a location of the niches)in mice, and the sensitivity to radiation carcinogenesis may be influenced by the irradiatedmicroenvironment. In skin (Annex F) there is clear evidence of a short hierarchical lineage,
and a model for human skin cancer proposed that stem cells were likely target cells for basal
cell carcinoma (BCC), early progenitor cells for squamous cell carcinoma (SCC), and lateprogenitor cells for papillomas. Concerning bone (Annex G), mesenchymal (stromal)/stem
cells (MSCs) for the osteoblast lineage reside in the bone marrow. CD34-negative stem cells
and mesenchymal precursors are possible target cells for radiation-induced bone cancers.
(l) Target cell location is an important consideration in the calculation of doses received
from short-range particulate emissions from radionuclides retained in body tissues, including
α particles and low energy electrons. The extent to which the immediate progeny of stem
cells may also be targets for the development of particular cancer types varies between tissues
and is not well established in many cases. For some tissues, this may not have implications
for the definition of targets for dosimetric purposes because stem cells and their immediate
progeny occupy the same microenvironment. Possible target cell locations are summarised inTable 3.2.
(m) In summary, answers to the questions posed in Chapter 1, Section 1.2, are as follows:• What are the target cells for carcinogenesis, and where are they located? In most cases
the target cells are the stem cells residing in the stem cell niche. In some cases also earlyprogenitor cells (daughter cells of the stem cells) may be target cells.
• Does the LNT model fit with considerations of stem-cell-based radiation carcinogenesis,
and if so, how? A single stem-cell origin of radiation-induced cancer and mutational
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theory are consistent with an LNT approach for some tissues and organs, albeit with the
caveat of the uncertain influence of radiation-induced epigenetic effects.
• Is the current DDREF value supported by information concerning stem-cell-based
radiation carcinogenesis? Suggested mechanisms, and evidence for their support (i.e.
stem cell competition and dose-rate dependent decrease in the slope of the linear term of
the LQ dose response curve) give less risk from chronic exposures than expected fromconsideration of solely the linear component. These mechanisms support a DREF value
greater than unity.
• What are the mechanisms related to the stem cell response, and do those mechanisms help
to explain the tissue differences in sensitivity to radiation carcinogenesis? Carcinogenesis
depends primarily on three mechanistic factors: (1) the number and sensitivity of stem
cells to radiation-induced mutation; (2) the retention of mutated stem cells in a tissue; and(3) the population size of stem cells with a sufficient number of predisposing mutations.
At present, there is a lack of definitive evidence for the various contributions of thesefactors to radiation carcinogenesis and hence to radiation risk in different tissues.
• What could be an underlying mechanism related to stem cells for the age-dependent
sensitivity to radiation carcinogenesis, and hence to risk? Stem cells exposed at the fetal
stage of development are less likely to be retained during neonatal growth, whereradiation-injured stem cells are under strong competition with unirradiated stem cells tosettle in the limited number of newly-established stem-cell niches. In contrast, highsensitivity in childhood can be understood if stem cell competition is less stringent,because the stem-cell/niches increase in number to cope with the increase in the tissue
volume during childhood growth.

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GLOSSARYα/β value or ratio
A measure of the curvature of the cell survival curve. The α/β is also the dose at which
the linear and quadratic components of cell killing are equal. For tissues, the α/β value isa measure of their sensitivity to changes in dose fractionation. In vivo, the α component
describes the dose-response slope at low doses, which is often considered independent of
dose-rate but likely it can be modified in chronic radiation scenarios by cell renewal andcell competition processes. The β component describes the increase in slope at higher
doses due to cumulative damage, which is repairable during fractionated or low-dose-rate
exposures.
Absolute risk (AR)The risk of an adverse health effect – the probability or rate of the occurrence of aparticular health event (e.g. disease incidence) over a specific period.
Absorbed dose, D
The energy imparted per unit mass by ionising radiation to matter at a specific point. The
SI unit for absorbed dose is joule per kilogram (J/kg) and its special name is gray (Gy).
Active (red) bone marrow
The organ system bone marrow contains the cell systems for the formation of blood cells
starting from the pluripotent haematopoietic stem cells (HSCs) to the mature blood cells.
Adaptive response
Increased resistance of cells or tissues to radiation following a priming dose, or
adjustment to radiation exposure which enables an organism to retain viability, maintainfertility and normal functional stability of all tissues, organs and systems under theconditions of chronic exposure. The principal criterion of radiation adaptation is anincreased radioresistance (tolerance) of the organism and the cells of its critical organs.
Adenoma
A benign tumour of glandular origin. Adenomas can grow from many organs including
the colon, adrenal glands, pituitary gland, thyroid, prostate, etc. Although these growths
are benign, over time they may progress to become malignant, at which point they arecalled adenocarcinomas (ADCs).
Apoptosis
A mode of cell death in which the cell nucleus displays characteristic densely staining
globules, and at least some of the DNA is subsequently broken down into
internucleosomal units. Sometimes postulated to be a ‘programmed’ and therefore apotentially controllable process.
Asymmetric division
Cell division producing two different types of daughter cells, e.g. a tissue stem cell
producing both a stem cell and a progenitor cell (see also Immortal strand hypothesis).

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Baseline disease rates
The annual disease incidence observed in a population in the absence of exposure to theagent under study.
Blastocyst
Structure formed in the early gestation of vertebrates. It is preceded by the morula. It
possesses an inner cell mass, or embryoblast, which subsequently forms the embryo, andan outer layer of cells, or trophoblast, surrounding the inner cell mass and a fluid-filledcavity known as the blastocoele. The human blastocyst comprises 70-100 cells.
Caretaker genes
Caretaker genes encode gene products that stabilise the genome.
Cell deathIn the context of radiobiology, cell death is generally equated with any process that leads
to the permanent loss of clonogenic capacity, often termed loss of reproductive integrity.
Cell death can also refer to physical death through a variety of processes such as
apoptosis, necrosis and autophagy, and also sometimes, premature senescence andpremature differentiation.
Checkpoint
A point in the cell cycle at which injured cells are arrested and then released after
recovery to progress to the next phase of the cell cycle.
Chromothripsis
Multiple genomic rearrangements with sharply circumscribed regions of one or a few
chromosomes, crisscrossing back and forth across involved regions.
Clonogenic cells
Cells that have the capacity to produce an expanding family of descendants (usually atleast 50). Also called ‘colony-forming cells’ or ‘clonogens’.
Clonogenic surviving fraction
The fraction of clonogenic cells that survive exposure to, or treatment with, an agent thatcauses cell death. Only cells that are able to form colonies (clonogenic cells) areconsidered to have survived the treatment (see Cell death).
CO-FISH
Chromosome orientation fluorescence in situ hybridisation technique. Can be used tocheck if the segregation of sister chromatids is random or not.
ColonyThe family of cells derived from a single clonogenic cell.
Complex tissues (also called flexible tissues or conditional renewing tissues)
Cell populations in which both function and proliferation can take place alternatively inthe same cells.
Confidence limits or intervals
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An interval giving the lowest and highest estimate of a parameter that is statisticallycompatible with the data. For a 95% confidence interval (CI), there is a 95% chance thatthe interval contains the parameter.
Cytokines
Organic molecules with biological function, originally defined as being polypeptides
released from lymphocytes and involved in maintenance of the immune system. These
factors have pleiotropic effects on not only haematopoietic cells but many other cell
types as well. Often synonymously termed growth factors.
Cumulative dose
The total absorbed dose resulting from repeated exposures to ionising radiation over a
period of time.
D0

A parameter in the multitarget equation for cell survival: the radiation dose that reduces
survival to e-1 (i.e. 0.37) of its previous value on the exponential portion of the survival
curve.

Dose and dose-rate effectiveness factor (DDREF)
A judged factor that generalises the usually lower biological effectiveness (per unit ofdose) of radiation exposures at low doses and low dose rates as compared with exposuresat high doses and high dose rates; includes dose effectiveness factor (DEF) and dose-rate
effectiveness factor (DREF).
Dose rate
The absorbed radiation dose delivered per unit time and measured, for example, in grayper hour.
Dose-rate effectDecreasing radiation response with decreasing radiation dose rate.
Elemental dose
The lowest dose given by a single track of radiation to a nucleus of a cell.
Embryonic Stem (ES) cells
Cells in the inner cell mass of the blastocysts, responsible for further development of theentire embryo proper.
Epigenetic effects
Epigenetic changes consist of changes in the properties of a cell that are inherited butthat do not represent a change in genetic information, e.g. methylation effects. They
influence the phenotype without alteration in the genotype.
Epithelium
Membranous tissue composed of one or more layers of cells, forming the covering ofmost external and internal surfaces of the body and its organs.
Erythropoietin
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Cytokine that regulates erythrocyte levels and stimulates late erythroid progenitor cells toform small colonies of erythrocytes.
Excess absolute risk (EAR)
The additional risk (or rate) from radiation exposure above the underlying (baseline) risk(or rate) of the disease. This is often expressed as the EAR per Gy or per Sv.
Excess relative risk (ERR)
The excess proportion (or percentage) of the rate of radiation-induced disease in an
exposed population divided by the rate of disease in an unexposed population which has
the same background risk factors (age, sex, race, etc.). This is often expressed as the
ERR per Gy or per Sv.
Exponential survival curve
A survival curve without a threshold or shoulder region, which is a straight line on asemi-logarithmic plot.
FACS
Fluorescence-activated cell sorting, which can be used to identify stem cells using
particular cell surface markers.
Fractionation and dose delivery patterns
The dose per fraction of radiation is the total dose divided into a particular number offractions. A very large number of extremely small dose fractions becomes equivalent to
low-dose-rate exposure. Very low dose-rates protracted over long durations are called
chronic exposures.
Fractionation sensitivity
The dependence of the isoeffective absorbed radiation dose on the dose per fraction.
Usually quantified by the α/β value – a high fractionation sensitivity is characterised by a
low α/β value (see α/β value).
γH2AX fociIdentification of the broken ends of DNA caused by ionising radiation. H2AX is one ofseveral genes coding for histone H2A. H2AX becomes phosphorylated on serine 139,then called γH2AX, as a reaction on DNA double-strand breaks (DSBs). γH2AX is asensitive target for looking at DSBs in cells.
Gatekeeper genes
Gatekeeper genes encode gene products that act to prevent growth of potential cancer
cells and prevent accumulation of mutations that directly lead to increased cellular
proliferation.
Genomic integrity
Preservation of the structural and functional content of the genome of cells.
Granulocyte colony-stimulating factor (G-CSF)
Cytokine that stimulates proliferation and differentiation of progenitor cells into
granulocytes.
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Granulocyte-macrophage colony-stimulating factor (GM-CSF)
Cytokine that stimulates proliferation and differentiation of progenitor cells into
granulocytes, macrophages, and eosinophils.
Gray (Gy)The special name for the SI unit of absorbed dose: 1 Gy = 1 J/kg.
Growth factor
An organic molecule which stimulates cell proliferation when it binds to its cell surface
receptor. Often synonymously termed cytokine.
Growth fraction
Proportion of viable cells in active cell proliferation.
Hierarchical tissues
Tissues comprising a lineage of stem cells, transit (amplifying) cells, and postmitotic(differentiated or mature) cells.
High linear energy transfer radiation
Radiation having a high linear energy transfer (LET), for example, α particles, heavy
ions and interaction productions of fast neutrons. The ionisation density along theradiation track is high.
Homologous recombination (HR)HR takes place in S and G2 phase cells to repair a damaged region of DNA by copying
the intact counterpart of the sister DNA strand. HR is potentially error-free.
Hypoplasia
Reduction in cell numbers in a tissue, e.g. owing to radiation-induced impairment ofproliferation in early-responding tissues.
Hypoxanthine-guanine phosphoribosyltransferase (Hprt) mutations
The Hprt assay is an in vitro mammalian cell gene mutation test. The estimation ofmutant frequency in the reporter gene, called Hprt located on the X chromosome, canprovide information on the biological effect of an absorbed dose in the cell type studied,and hence is a useful biodosimetry tool.
Immortal strand hypothesis
Asymmetric segregation of DNA strands to minimise the replication error in the stem
cells. The stem cell retains the template DNA strand after a round of DNA synthesis,
while the progenitor cells inherit the daughter strand.
Incidence (incidence rate)
The rate of occurrence of a disease in a population within a specified period of time,often expressed as the number of cases of a disease arising per 100,000 individuals per
year, or per 100,000 person-years (PY).
Initial slope
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The steepness of the initial part of the cell survival curve, usually indicated by the valueof α in the linear-quadratic (LQ) model.
Interphase death
The death of irradiated cells before they reach mitosis. Sometimes used as a synonym for
apoptosis.
Knockout miceMice in which one (or more) gene has been inactivated.
Label-retaining cells (LRCs)
Cells which retain a DNA label through multiple rounds of cell division.
Lifetime risk
The cumulated risk of morbidity or dying of some particular cause up to a given age.
Linear energy transfer (LET)The rate of energy loss along the track of an ionising particle, usually expressed in
keV/µm.
Linear-no-threshold (LNT) dose-response model
A dose-response model which is based on the assumption that, in the low dose range, any
radiation doses greater than zero will increase the risk of excess cancer and/or heritabledisease in a simple proportionate manner.
Linear-quadratic (LQ) dose-response modelA statistical model that expresses the risk of an effect E (e.g. disease, death, orabnormality) as the sum of two components, one proportional to dose (linear term) andthe other one proportional to the square of dose (quadratic term). E = αD + βD2, where D
is dose. For cell survival: S = exp - (αD + βD2).
Low-LET radiation
Radiation having a low LET, for example electrons, x-rays, and γ-rays.
Lymphatic system
A network of lymphatic vessels of varying calibre that collects tissue fluids from all overthe body and returns these fluids to the blood. Accumulations of lymphocytes, called
lymph nodes, are situated along the course of lymphatic vessels.
Macrophage colony-stimulating factor (M-CSF)
Cytokine that stimulates formation of macrophages from pluripotent haematopoietic
cells.
MammosphereA spheroid of cells derived from single mammary gland cells. A single cell from a
mammosphere can regenerate an entire mammary gland when transplanted into a
mammary fat pad.
Multiple intestinal neoplasia (Min) mouse17

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Min mice are genetically heterozygous for a germ-line truncating mutation of the
adenomatous polyposis coli (Apc) gene (i.e. ApcMin/+), and develop multiple intestinal
tumours and sporadic colon tumours in their intestinal tracts within several weeks ofbirth. The Min mouse provides a sensitive model for the study of tumourigenesis in
irradiated mice.
Multistage carcinogenesis model
Carcinogenesis model associated with a stepwise acquisition of mutations of oncogenes
and tumour suppressor genes, associated with a progressive loss of external proliferative
factors.
Necrosis
Cell death associated with loss of cellular membrane integrity. Occurs for example inanoxic areas of tumours and is also a cause of cell death after irradiation.
Never-smokers
People who have never smoked.
Niche (or stem cell niche)
Specific microenvironment in a tissue where stem cells reside and are maintaind byvarious signals controlling proliferation and differentiation.
Non-homologous end joining (NHEJ)
NHEJ repair takes place in non-cycling cells and variously in all cycle phases, and isdependent on the repair proteins Ku70, Ku80 and DNA-dependent protein kinasecatalytic subunit (DNA-PKcs).
Non-smokers
People who do not smoke.
Non-targeted effects
Indirect effects of radiation exposure, including bystander effects and the induction of
genomic instability.
Oncogene
A gene that when mutated or overexpressed, contributes to converting a normal cell intoa cancer cell.
Platelet-derived growth factor (PDGF)
A cytokine which induces growth of fibroblasts and is involved in wound healing. Also
acts on some epithelial and endothelial cells, and on mesenchymal cells.
Poisson distribution
Distribution applicable when the probability of an event happening is small but the
number of observations is large. The distribution of probabilities runs from zero to
infinity, and an important characteristic of the distribution is that the mean equals thevariance.
Potentially-lethal damage repair (PLDR)
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DNA repair occurring in a delay period after irradiation, before cell division occurs.
Programmed cell death
Cell death that occurs as the result of an active process carried out by molecules in thecell. Examples include apoptosis, autophagy, terminal differentiation, senescence, and insome cases even necrosis.
Protection quantities
Dose quantities that the Commission has developed for radiological protection, thatallow quantification of the extent of exposure of the human body to ionising radiationfrom both whole and partial body external irradiation and from intakes of radionuclides.
RadioresponsivenessTemporal rate of response of a tissue to irradiation (in contrast to the magnitude of
response, i.e. radiation tolerance). This depends on multiple factors, one of themhypothesised to be cellular radiosensitivity.
Radiosensitiser
In general, any agent that increases the sensitivity of cells and tissues to radiation.
Commonly applied to electron-affinic chemicals that mimic oxygen in fixing free-radical
damage, although these should more correctly be referred to as hypoxic cell sensitisers.
Radiosensitivity, cellular
The sensitivity of cells to surviving exposure to ionising radiation. Usually indicated by
the surviving fraction at 2 Gy (i.e. SF2) or by the parameters of the LQ or multitarget
equations.
Reactive oxygen species (ROS)
Molecular species such as superoxide, hydrogen peroxide, and hydroxyl radicals. These
species may function in cell signalling processes. At higher levels, these species may
damage cellular macromolecules (such as DNA and RNA) and participate in cell deathprocesses.
RecoveryAt the cellular level: an increase in cell survival as a function of time between dose
fractions or during irradiation at low dose rates. At the tissue level: an increase in tissueequi-effective total dose with an increase in time interval between fractions and adecrease in dose per fraction, or with irradiation at low dose rates.
Relative biological effectiveness (RBE)The ratio of a dose of a low-LET reference radiation (usually of 60Co γ−rays or
kilovoltage x-ray quality) to a dose of the test radiation considered that gives an identicalbiological effect. RBE values vary with the dose, dose fractionation, dose rate, and abiological endpoint considered.
Relative risk (RR)
An expression of overall risk (i.e. including the radiation-induced risk) relative to theunderlying baseline risk. If the total risk is twice the underlying baseline risk then the RR
is 2.
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Reproductive cellular integrity
Ability of cells to divide many times (usually >5) and thus be ‘clonogenic’.
Senescence
A permanent arrest of cell proliferation associated with differentiation, ageing, or cellular
damage.
Side population (SP)In flow cytometry, a subpopulation of cells that is distinct from the main population on
the basis of the markers employed - often cells that show higher efflux of DNA-bindingdye Hoechst 33342. By definition, cells in a side population have distinguishingbiological characteristics (for example, they may exhibit stem cell-like characteristics),
but the exact nature of this distinction depends on the markers used in identifying theside population.
Sievert (Sv)
The special name for the SI unit of equivalent dose, effective dose, and operational dosequantities in radiation protection. The unit is joule per kilogram (J/kg).
SKY analysis
Spectral karyotyping (SKY) of the chromosomal content of cells using metaphase cells
pretreated and hybridised with a SKY probe mixture containing uniquely labelledchromosome-specific probes.
Slow repair
Long-term restoration of radiation tolerance that takes place on a time scale of weeks toyears, often associated with long-term intracellular repair.
Spheroids
Aggregates of cells produced in culture by multiple divisions of a single cell. Spheroids
can be produced both from some normal and malignant cells, the latter being often used
as a model of tumour metastases.
Stem cells
Cells with an unlimited proliferative capacity, capable of self-renewal and ofdifferentiation to produce all the various types of cells in a lineage system. Stem cells aredescribed as being totipotent (producing all lineages), whereas their daughter progenitor
cells can be multipotent (producing several lineages) or unipotent (one lineage).
Stemness
Stem cell characteristics that underlie self-renewal and the ability to generate
differentiated progeny. There are differing degrees of stemness among more primitiveand less primitive stem cells in some hierarchical lineage systems.
Stochastic effects of radiation
Malignant disease or heritable effects; the probability of an effect occurring, but not its
severity, is regarded as a function of dose without threshold.

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Sublethal damage repair (SLDR)DNA repair occurring during low-dose-rate exposure or between dose fractions, resulting
in less cell kill or less tissue reaction than if the total dose was delivered acutely.
Targeted effects
Effects occurring in irradiated cells.
Telomeres
The ends of chromosomes. One of the determinants of cellular senescence is the loss oftelomeres through DNA replication. Rapidly replicating cells usually have telomerase
activity to avoid telomere shortening. Shortening of telomeres is also associated withgenomic instability and carcinogenesis.
Teratocarcinoma
A malignant neoplasm consisting of elements of teratoma with those of embryonalcarcinoma or choriocarcinoma, or both; occurring most often in the testis.
Tissue weighting factor, wT
The factor by which the equivalent dose in a tissue or organ T is weighted to represent
the relative contribution of that tissue or organ to the total health detriment resulting from
uniform irradiation of the body.
Transforming growth factor beta (TGFβ)
A cytokine that regulates many of the biological processes essential for embryo
development and tissue homeostasis, and which therefore plays a role in the healing ofsome tissues. The effects of TGFβ may differ depending on the tissue involved, e.g.
TGFβ inhibits the proliferation of epithelial cells but stimulates proliferation,differentiation and collagen synthesis in fibroblasts.
Transit cells
Differentiating proliferative cells that amplify cell production in a hierarchical tissue.
Translocations
Chromosomal abnormalities which occur when chromosomes break and the fragments
rejoin to other chromosomes. There are many structurally different types oftranslocations.
Trophectoderm
The outer layer of the mammalian blastocyst after differentiation of the ectoderm,
mesoderm, and endoderm, when the outer layer is continuous with the ectoderm of theembryo.
Tumour suppressor gene
A tumour suppressor gene, or anti-oncogene, is a gene that protects a cell from one stepon the path to cancer. When this gene is mutated to cause a loss or reduction in its
function, the cell can progress to cancer, usually in combination with other genetic
changes.
Working level month (WLM)
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The cumulative exposure from breathing an atmosphere at a concentration of 1 Working
Level (WL) for a working month of 170 hours. In the case of radon, 1WL = any
combination of the short-lived progeny of radon in 1 litre of air that will result in theemission of 1.3x105 MeV of potential alpha energy. 1 WL = 2.08x105 J/m3.
Main reference source for the Glossary
ICRP, 2012. ICRP Statement on Tissue Reactions / Early and Late Effects of Radiation in Normal
Tissues and Organs – Threshold Doses for Tissue Reactions in a Radiation Protection Context.
ICRP Publication 118. Ann. ICRP 41(1/2).

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ABBREVIATIONS
53BP1
ADC
ALL
AML
Ang-1
AR
ATM
BASC
BCC
Bcrp1
bFGF
BrdU
CBCC
CFU-F
CML
CMP
CNS
DDREF
DEF
DREF
DSB
EAR
EGF
EpiSC
ERR
ES
FACS
FAP
FISH
G-CSF
GFP
GM-CSF
GMP
Hprt
HR
HSC
HSPC
IGF1
Klf4
Lgr5
LNT
LQ
LRC
MAPK
MaSC
M-CSF

p53-binding protein 1
adenocarcinoma
acute lymphoblastic leukaemia
acute myeloid leukaemia
Tie2/angiopoietin 1
additive risk
ataxia telangiectasia mutatedbronchioalveolar stem cellbasal cell carcinoma
breakpoint cluster region pseudogene 1
basic fibroblast growth factor
bromodeoxyuridine
crypt base columnar cells
fibroblastoid colony-forming unit
chronic myeloid leukaemia
common myeloid progenitor
central nervous systemdose and dose-rate effectiveness factor
dose effectiveness factor
dose-rate effectiveness factor
double strand breakexcess absolute risk
epidermal growth factor
epidermal stem cell
excess relative risk
embryonic stem
fluorescence-activated cell sorting
familial adenomatous polyposis
fluorescent in situ hybridisation
granulocyte colony-stimulating factor
green fluorescent protein
granulocyte-macrophage colony-stimulating factor
granulocyte/macrophage progenitor
hypoxanthine-guanine phosphoribosyltransferase
homologous recombinationhaematopoietic stem cell
haematopoietic stem and progenitor cell
insulin-like growth factor 1
Kruppel-like factor 4
leucine-rich repeat-containing G protein-coupled receptor 5
linear no thresholdlinear quadraticlabel-retaining cell
mitogen-activated protein kinase
mammary gland stem cells
macrophage colony-stimulating factor
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985	
  
986	
  
987	
  
988	
  
989	
  
990	
  
991	
  
992	
  
993	
  
994	
  
995	
  
996	
  
997	
  

Min
miRNA
Mre11
MSC
mTert
NHEJ
NSC
Oct3/4
OSCC
PDGF
PLDR
RBE
ROS
RR
SCA-1
Sca-1
SCC
SCF
SCGBa1a
SCLC
SCN
SI
SLDR
Sox2
SP
TA
TGFβ
WLM

multiple intestinal neoplasia
microRNA (microribonucleic acid)meiotic recombination 11
mesenchymal (stromal)/stem cellmouse telomerase reverse transcriptase
non-homologous end joining
neural stem celloctamer-binding transcription factors 3/4
Oxford Survey of Childhood Cancers
platelet-derived growth factor
potentially-lethal damage repair
relative biological effectivenessreactive oxygen species
relative riskstem cell antigen-1
type II cell marker surfactant protein C
squamous cell carcinoma
c-kit tyrosine kinase receptor
secretoglobin a1asmall-cell lung carcinoma
solid cell nest
small intestine
sublethal damage repair
sex determining region Y-box 2
side populationtransit amplifying
transforming growth factor beta
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1. INTRODUCTION

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1.1. Purpose of the report

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(1) The risks of radiation-induced cancer have continued to dominate the reasons behindrecommendations on restricting radiation exposures of workers and the public for many
decades. Carcinogenesis from radiation is considered to be a stochastic event, originating in asingle transformed target cell. Generally, the target cells are considered to be the stem cells
and possibly also some of their daughter progenitor cells in each tissue. These target cells
have tissue-specific characteristics, and they reside in a microenvironmental “niche” that
regulates their proliferation and differentiation (see Fig. 2.1.). Knowledge about the stem
cells and their regulation may help underpin extrapolated risk estimates for different tissue
and organ systems, and may also help understand risk projections in different exposure
scenarios.
(2) The location of target cells in different tissues is vitally important regarding the risksof carcinogenesis from short-range radionuclides and lightly-penetrating radiation beams. In
its Publications, the Commission has made some judgments and assumptions about thelocation of these cells. In the skin, the target cells are considered to be in the hair follicles aswell as in the basal interfollicular epidermis (ICRP, 1991). In the respiratory tract, the target
cells are considered to be in the basal layer of the mucosa and in the alveoli of the lung (ICRP,
1995). In the intestine, the target cells are considered to be near the bottom of the intestinal
crypts, but there are uncertainties about the carcinogenic potential of cells further up the
crypts (ICRP, 2007). In the skeleton, the target cells responsible for radiation-induced
osteosarcomas are considered to be the osteoblasts in bone cavities, as well as mesenchymal
stem cells (MSCs) in the bone marrow (ICRP, 1996). The information in the present report is
likely to contribute further to clarification of the target cell locations in the tissue of interest.
(3) The report comprises a review of advances in knowledge of the biology and radiation
response of stem cells and progenitor cells in the context of the tissue microenvironment, in
relation to mechanisms of radiation carcinogenesis. Recent progress in stem cell biology andradiobiology is described, including tissue architecture, the dynamic nature of tissue
maintenance, stem cell radiosensitivity and renewal, dose-rate effects and age dependence.
This information is then evaluated for deducing the implications of the role of stem cells in
the mechanisms of carcinogenic risk as a function of acute and chronic radiation dose,
including projections of risk for short-range radiations.
(4) In order to form a basis of scientific knowledge regarding stochastic radiation effects
in different tissues and organs, a series of organ systems was selected as examples withknown radiation-induced risks. As such reviews are not available currently, a detailed seriesof Annexes was produced for these individual examples. Not all organ systems were covered
because of the enormity of such a task. Selection was made on the basis of importance forradiation protection purposes and of the extent of available radiobiological knowledge andinterest. Firstly, those with the highest ICRP tissue weighting factor of 0.12 were selected,
which include bone marrow, breast, digestive tract and lung. Among these, haematopoietic
tissue is best studied for the stem cell aspect (Annex A), and there is good knowledge of stem
cell locations and other hierarchical aspects of tissue turnover in the digestive tract (Annex D).
The presence of stem cells is well proven for the mammary gland (Annex B), but those of thelung have yet to be fully characterised (Annex E). Thyroid was selected for its strong age
dependence of susceptibility to radiation carcinogenesis (Annex C). Bone was selected
because it is the best model system for cancer induction by internal emitters (Annex G). Skin

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was selected for its simple hierarchical tissue structure which facilitates the understanding
regarding tissue cell types, cancer sub-types and their susceptibility to radiation (Annex F),
and the large, but rather uncertain, skin cancer incidence rate (ICRP, 1991, 1992, 2007;
Annex F). The format of the Annexes was designed so as to present similar information foreach tissue, in order to facilitate comparisons.

1050	
  

1.2. Radiation carcinogenesis models and stem cell biology

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(5) Current understanding of mechanisms for radiation-induced cancer relies on themultistep model of Armitage and Doll (1954), and its extension to the molecular changes
postulated by Vogelstein et al. (1988). However, these mechanisms have limited impacts
since they were not thoroughly evaluated in the context of radiation carcinogenesis.
Improvements in this situation can be attained by positioning the LNT and RR models in thecontext of the multistep model. Improvements in this situation may arise from theidentification of target cells within tissues, understanding of radiation responses of these cells,
and the kinetics of turnover and cell-cell interactions that control the potential of these target
cells to progress in the carcinogenic process. These issues are important for external
exposures particularly for low chronic exposures but they take on added importance when
considering risks from internal emitters, particularly those with short ranges in tissues. The
newly emerging stem cell biology provides a real opportunity to clarify these issues andprovide a foundation to better understand the dose and dose-rate effects and better define thetarget-cell locations for dose effects from internal emitters. It may also contribute to reasonssupporting the way to transport risks across different populations.
(6) The number, sensitivity, location and renewal characteristics of target cells areimportant biological parameters with respect to carcinogenesis from radiation. Many people
consider that the target cells for carcinogenesis are the tissue stem cells and some of theirdaughter cells. The number of stem cells is often ill-defined and estimated using various
assays in different tissues in experimental animal systems. Stem cell radiosensitivity varies
both within and among tissues. For example, there are both resistant slow-cycling andsensitive fast-cycling stem cells in spermatogenic epithelium, sensitive apoptosis-susceptible
cells and more resistant clonogenic cells in intestinal mucosa, and more sensitive stem cells
in haematopoietic than in epithelial tissues. The microenvironment within tissues differs in avariety of parameters including cell-to-cell and cytokine-mediated signalling.
(7) The location of target cells in different tissues is vitally important regarding the risksof carcinogenesis following radiation doses from short-range radionuclides and lightlypenetrating radiation beams. Stem cells are often found residing in a specificmicroenvironment known as a stem cell “niche”. The target cells for leukaemia areconsidered to be HSCs and possibly some of their daughter cells, and their niches are situatedwithin small bone cavities as well as near sinusoids. The density of some stem cell types near
the central blood vessel is lower than closer to the bone surface, although there is someevidence that the reverse is true regarding their renewal ability. However, the general
conclusion is that the primary stem cells are protected in hypoxic niches near the bone
surface (Parmar et al., 2007). These uncertainties in target cell type and position have
hampered attempts to provide accurate projections of radiation risks pertaining to short-range
radiations, based on risks for homogeneous external irradiations. Moreover, the stem cellniche and its morphological features are still to be better defined for many tissue types suchas mammary gland and thyroid.
(8) For purposes of radiological protection programmes, the ICRP applied the LNT modelto the risk assessment of chronic exposures, with the use of a DDREF. The numerical value

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of DDREF remains controversial, and various proposals have been made (BEIR VII, 2006).
Evaluation of stem cells and tissue turnover is expected to help clarify the appropriateness ofusing LNT for low dose extrapolation and a DDREF value for adjustment of risk for chronic
exposures. The latter is dependent to some extent on the uncertain repair capacity and therenewal/retention rate of the target cells in tissues. Although DNA repair has been discussed
regarding its role in the DDREF, the cell renewal rate is considered to be more important forcancer types where the target cells are not only the stem cells, but also their daughterprogenitor cells, which are subject to more vigorous renewal. In addition, recent studies haveshown that even the damaged tissue stem cells in some cases are subject to constant removal
competition from undamaged stem cells. This would be expected to result in a lower risk
from chronic exposures than that predicted simply by the cumulative dose to the initial
complement of target cells, which are being slowly replaced by new cells.
(9) The following chapters discuss the following questions and topics in the light ofrecent progress in stem cell biology: What are the target cells for carcinogenesis, and where
are they located? Does the LNT model fit with considerations of stem-cell-based radiation
carcinogenesis, and if so, how? Is the current DDREF value supported by information
concerning stem-cell-based radiation carcinogenesis? What are the mechanisms related tostem cell response, and do those mechanisms help to explain the tissue differences insensitivity to radiation carcinogenesis? What could be an underlying mechanism related tostem cells for the age-dependent sensitivity to radiation carcinogenesis, and hence risk?

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2. GENERAL FEATURES OF TISSUE STEM CELLS

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2.1. Cell division and differentiation in adult tissues

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(10) Adult tissues fulfill various bodily needs with specialised sets of functional cells.
Functional cells are terminally differentiated cells with a limited, often no, capacity to
proliferate and they have to be replaced when needed. Adult tissues can be divided into two
types, complex (flexible) and hierarchical types. In the flexible tissue such as liver, thefunctional cells have a potential to divide and can increase their number in special occasions
especially when injured, but they are thought to be supplied by stem cells under normal
conditions. In contrast, the turnover rate of the hierarchical tissues is high and functional cells
in this tissue type are lost rapidly from the body. For the supply of a large number of a variety
of functional cell types, the hierarchical tissues have a discrete lineage consisting of stem
cells, progenitor cells and differentiated cells. The hierarchical tissues are the main target forradiation carcinogenesis and they are the focus of the present report.
(11) Stem cells differ between early embryogenesis, fetal development and after
establishment of adult tissues. The stem cells of the embryonic stage are ES cells which are
totipotent and have the capacity to differentiate into all tissue types of later organogenesis. In
the fetal stage, stem cells are lineage-committed to a certain extent in order to contribute tospecific tissues of the adult stages. In these two stages of early life, stem cells mostly undergo
symmetric division to produce two equal daughter stem cells, associated with an increase in
the size of the embryo and the fetus (see Section 2.3.5 and Fig. 2.5.). By contrast, adult tissue
stem cells are mainly fully committed with restricted differentiation capabilities, and they
divide in an asymmetric fashion, although they undergo symmetric division to various extents
especially when repairing tissue injuries.
(12) The study of adult tissue stem cells started as early as 1960s. Haematopoietic
progenitor cells (originally considered as stem cells) were first identified in bone marrow as
those cells capable of forming splenic colonies after intravenous injection into lethallyirradiated mice (Till and McCulloch, 1961). Those early studies focussed on the regenerative
capacity of tissue progenitor cells, but later studies in vivo revealed many importantcharacteristics of tissue stem and progenitor cells including asymmetrical division (Potten
and Loeffler, 1990).
(13) Asymmetric division of adult tissue stem-cells produces both a stem cell and aprogenitor cell. Progenitor cells are also called transit amplifying (TA) cells, especially for
epithelial tissues. Although the terms of progenitor cells and TA cells are used somewhat
differently in the context of the haematopoietic system, these two terms are usedinterchangeably in the present report. The progenitor cells divide further to increase in
number and they then differentiate into functional cells which are eventually lost bysenescence from the tissue after serving their required functions. In this general scheme oftissue turnover, the stem cells are frequently quiescent while progenitors divide more rapidly
with a limited proliferative capacity. This differential role sharing between tissue stem cells
and progenitor cells is the strategy for life-long preservation of stem cells by minimising
replication-mediated mutations while supplying a large number of cells to the functional
compartment of a tissue by vigorous division of progenitor cells. The progression from stem
cells to differentiated cells is usually unidirectional, but can be reversible under certain
conditions such as when stem cells are lost for some reason where the vacant stem cell niche
becomes occupied by a neighbouring stem cell or by a dedifferentiated progenitor cell. The
latter scenario was demonstrated for germ cells of fruit flies and mice (Cheng et al., 2008;28

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Barrocal et al., 2009). The balance between the production and loss of cells maintains thebodily tissue mass.
(14) Hierarchical tissues contain three cellular compartments: the stem cellcompartment; the progenitor cell compartment; and the functional cell compartment. The
cells in the former two compartments have a capacity to divide while those in the lastcompartment generally do not. The steps from stem cells to the differentiated cells vary fromtissue to tissue. This somewhat oversimplified scheme is depicted in Fig. 2.1. Importantly,
while each cell resides in a defined compartment, the population as a whole consists of agenerally unidirectional gradient of cells between compartments.

Fig. 2.1. Stem cell division and the maintenance of tissue dynamics. A niche is the location for stem
cells, which divide to replace themselves or to produce progenitor cells. The latter divide further,producing functional mature cells. The functional cells have a limited lifespan before they die,requiring replacement by more divisions in the cell lineage.

(15) The number of cell stages in a lineage in a tissue varies greatly. In some tissues,stem cells supply the relatively limited number of lineages as in the case of the epidermis,
while those in other tissues supply a variety of lineages as exemplified by HSCs in bonemarrow. In addition, the number of divisions from the stem cells to functional cells isvariable among tissues as schematised in Fig. 2.2. (Potten and Wilson, 2007). As mentioned
earlier, differentiated cells are capable of cell division in flexible tissues such as liver, thyroid
and lung when injured. (permission needed)

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Fig. 2.2. A diagrammatic representation of a stem-cell-derived cell lineage. The number of cell
generations in the lineage varies considerably between tissues, with stratified and glandular epithelia
having the fewest amplifying divisions of dividing transit cells. For example, in the large-intestinal
crypt, there are 5-9 amplifying cell divisions. Seven divisions would produce 128 post-mitotic
maturing cells from 64 cells that had multiplied (proliferative cells) from one stem cell. Hence thepercentage of stem cells in the proliferative cells in this example would be 1/65 x 100 = 1.5 %. In
breast or tongue epithelium the lineage is short with likely only 1-2 amplifying cell divisions. Two
divisions would produce 4 maturing cells from 2 proliferative cells arising from 1 stem cell. Hence thestem cells form 1/3 x 100 = 33 % of the proliferative cells (Diagram reproduced from Potten andWilson, 2007). (Permission needed)(16) The rate of turnover varies markedly between tissues. The 14C dating technnique
was used to estimate directly the rate of cell turnover in various tissues and demonstrated thatthe majority of cells in the brain stays for the entire life span of individuals without being
replaced, while those in the blood and intestine turn over rapidly (Spalding et al., 2005).
Since the technnique measures the level of 14C isotope of all the cells in a tissue, it is notsensitive enough to exclude the possible existence of small number of cells which turn over.
In fact, stem cells are known to be present in adult brain (Quinones-Hinojosa et al., 2007), but
their contribution to the total turnover of brain tissue is too small to be detected by thistechnique.
(17) The same technology demonstrated the turnover rate of the fat cells in human to be
10% annually (Spalding et al., 2008). The turnover rate varied with age, and in
cardiomyocytes it decreased from 1% annually at the age of 25 to 0.45% at the age of 75
years (Bergmann et al., 2009). The turnover rate and the number and the location of various
cell types in a tissue are believed to be important determinants of tissue-specific risk ofradiation carcinogenesis by external exposures as well as by internal exposures. In adulttissues, hierarchical tissues with higher renewal rates are generally more sensitive to radiation

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carcinogenesis (small intestine is an important exception) than flexible/complex tissues withlower renewal rates. Also, the turnover rate varies by the developmental stage and age ofindividuals, and is one of the major determinants of the age dependence of radiation
carcinogenesis (see Sections 2.5.4 and 3.6).

1218	
  

2.2. Functional identification and isolation of tissue stem cells

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(18) Tissue stem cells are defined by their ability to self-renew and to produce progenitor
cells of particular lineages, which in turn give rise to all the cells of the tissue. The serialtransplantation of marrow into irradiated mice demonstrates that the cells in the colonies havea capacity to self-renew. Also, histological examination demonstrates the presence of several
cell types in a colony, indicating that the transplanted colony-forming cells have the capacity
to differentiate into various haematopoietic cell lineages. In addition, colony survival
analyses enable the estimation of radiosensitivity of the bone marrow stem/progenitor cells.
Studies of total bone marrow transplantation in combination with limited dilution of the cell
preparation estimated the fraction of the stem cells to be 1/10,000 to 1/100,000 (Harrison etal., 1993; Szilvassy et al., 1990).
(19) The functional identification of stem/progenitor cells by in vivo colony formation
was successfully applied for other tissues including the skin epidermis and intestinal
epithelium (Withers, 1967; Withers and Elkind, 1969). Transplantation of mono-dispersed
cells and tissue fragments was also used to demonstrate the presence of stem cells in mouseand rat mammary glands, and in rat thyroid (Daniel et al., 1971; Clifton et al., 1986). The
colony assay was used also for quantifying the radiation induction of carcinogenic events in
rat mammary and thyroid clonogenic cells (Kamiya et al., 1995; Watanabe et al., 1988).
Although these in vivo studies identified stem cells by their functions, lack of isolated stem
cells hampered further studies on the nature of the cells.
2.2.1. Isolation of embryonic and adult tissue stem cells(20) ES cells were established by explant culture of mouse blastocysts in vitro (Evans
and Kaufman, 1981; Martin, 1981). There are 20-40 ES cells in the inner cell mass of theblastocysts and they are responsible for further development of the entire embryo proper. ES
cells can be identified by their characteristic colony morphology in vitro of small and tightlypacked cells. These cells are immortal and express telomerase to protect their chromosome
ends (Carpenter et al., 2003). Also they are totipotent since ES cells contribute to almost all
tissues, except for the trophectoderm. This totipotency was shared by embryonic carcinoma
cells and early stage embryos, and ES cells readily form teratocarcinomas when transplanted
into allogenic sites (Rossant and Papaioannou, 1984).
(21) ES cells have been isolated from a variety of mammalian species including humans(Thomson et al., 1998). ES cells serve as the target of gene ablation techniques and areplaying a pivotal role in creating ‘knockout’ mice, thus contributing to the study of genefunctions in the tissue and whole-body contexts. A variety of repair-gene knockout mice has
been created which offers great opportunities for the functional analyses of these genes interms of radiosensitivity, DNA repair, mutagenesis and carcinogenesis in mice (Griffin et al.,2005; Zha et al., 2007). In subsequent sections of this report, however, ES cells are discussed
only when necessary, because they have no direct relevance to radiation carcinogenesis.
(22) Isolation and in vitro cultivation of cells facilitate qualitative and quantitative
analyses of tissue stem cells. Long term in vitro cultivation of tissue stem cells was
accomplished for mouse haematopoietic cells in 1976, but only to produce granulocytic cells

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(Allen and Dexter, 1976). Numerous attempts have been made since then which resulted in
identification of various cytokines for the growth and differentiation of HSCs. HSCs and theirprogenitors now can be maintained in a defined culture medium in the presence of cytokines(Miller and Eaves, 1997). However, the degree of the expansion of HSCs under in vitro
cultivation is still modest, while such expansion through serial transplantation in irradiatedmice was shown to be more than 8000-fold (Iscove and Nawa, 1997; Sauvageau et al., 2004).
(23) Isolation and in vitro cultivation of tissue stem cells are now greatly facilitated by
identificationofvariousstem
cell-specificmarkerproteins(http://stemcells.nih.gov/info/scireport/appendixe). Among these, cell-surface marker
proteins are particularly useful to isolate tissue stem cells by fluorescence-activated cellsorting (FACS) (Gundry et al., 2008). FACS sorting of HSCs relies on specific cell surface
markers, such as c-kit tyrosine kinase receptor (SCF), stem cell antigen-1 (SCA-1) and CD34
(Shizuru et al., 2005). Cell surface markers (Table 3.2.) are described in the Annexes fortissue specific stem cells.
(24) In addition to the cell surface markers, a unique cellular property of the side
population (SP) phenotype is shared by many tissue stem cells. This was exploited to enrichand isolate stem cells using flow cytometry. When bone marrow cells are stained withfluorescence dyes of Rhodamine 123 and Hoechst 33342, the most-weakly-stained fraction isfound to contain long-term HSCs. This weak staining is associated with the low metabolicand mitotic activities of quiescent HSCs (Bertoncello and Williams, 2004). A high expressionof the ABC transporter, breakpoint cluster region psueudogene 1 (Bcrp1), and the resulting
efficient efflux of the dye are responsible for the SP phenotype of the quiescent HSCs (Zhouet al., 2001).
(25) Isolation and cultivation of stem cells were also accomplished by exploiting anotherunique feature. Neural stem cells (NSCs) and mammary gland stem cells (MaSCs) exhibit the
SP phenotype as in HSCs, but in addition, they form spheroids when cultured in vitro (Annex
B). A majority of cells died when a single cell suspension of the periventricular region of theadult mouse brain was cultured in a medium supplemented with epidermal growth factor(EGF). However, a small population of cells (about 1%) grew and formed spheroids(Reynolds and Weiss, 1992). Neurospheres, as they were called, were enriched with NSCs
and their progenitor cells. Neurosphere formation is a valuable tool for quantitative
assessment of radiation effects on the neural cells and such assay has been conducted on rat
spinal cord stem cells (Lu and Wong, 2005). Spheroid formation was also noted for human
MaSCs cultivated in the presence of EGF and/or basic fibroblast growth factor (bFGF)(Dontu et al., 2003). As is the case for the neurospheres, mammospheres also can be seriallypassaged. In addition, a single cell from a mammosphere can regenerate an entire mammary
gland when transplanted into a mammary fat pad (Shackleton et al., 2006). In vitro
cultivation and mammosphere formation offer a great opportunity in analyzing radioresponse
and radiosensitivity of MaSCs, considering that mammary gland is one of the highly
susceptible tissues for radiation carcinogenesis.
(26) HSCs originate from endodermal tissues, and NSCs and MaSCs from ectodermal
tissues (Annex A). In addition, MSCs can be propagated successfully in vitro (Chamberlain
et al., 2007). Thus, stem cells are expected to be isolatable from almost all of the tissue types.
One problem of the current systems of FACS-mediated isolation and in vitro cultivation ofstem cells is that the propagated population, whilst enriched for stem cells, still contains theirdescendants. Isolation of a pure stem cell population is yet to be accomplished.
(27) Recent advances of stem cell research have demonstrated a hierarchy of stem cells,
especially for tissues of rapid turnover rate. For example, stem cells of the haematopoietic
system can be classified into at least long-term HSCs and short-term HSCs, the former being
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more primitive than the latter (Annex A). As for the small intestine, three types of stem cells
have been identified within the various cell populations present in murine crypts (Annex D).
There are two distinct stem cell populations located at the 4th position from the crypt base,which are highly apoptosis-sensitive P4 stem cells and highly-radioresistant stem cells withmouse telomerase reverse transcriptase (mTert) expression. The third type of stem cells are
rapidly-cycling columnar cells at the crypt base, which are positive with leucine-rich repeatcontaining G protein-coupled receptor 5 (Lgr5). In the lung, there are region-specific stem
cells (Annex E); bronchioalveolar stem cells (BASCs), Clara cells and Clara variant cells
whose hierarchical interplay needs to be further clarified. Human skin seems to have a moreclear structure of epidermal stem cells (EpiSCs), early progenitors and late progenitors, eachgiving rise respectively to basal cell carcinoma (BCC), squamous cell carcinoma (SCC) andpapilloma (Annex F).

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2.3. Radiosensitivity and DNA damage response of tissue stem cells

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2.3.1. Basic strategies of tissue stem cells for the maintenance of genomic integrity(28) Current knowledge on DNA damage response pathways was summarised by ICRP
in Publication 99 (2005). DNA damage response is particularly important for maintaining the
genomic integrity of stem cells, since they have to supplement all the functional cells in atissue throughout life. This can be achieved by minimising DNA damage, cell divisions andDNA replication, maximising DNA damage repair, and eliminating damaged/mutated cells.
Indeed, failures of DNA damage response and DNA repair have been implicated repeatedly
in human premature ageing syndromes, many of which can be viewed as the consequence ofa premature exhaustion of tissue stem cells, and human cancer-prone syndromes (Friedberg etal., 2006). Abundance of antioxidants in stem cells and the provision of a hypoxic
microenvironment by the stem cell niche contribute to minimising DNA damage for sometissue stem cells. Quiescence is a feature shared by stem cells of a variety of tissues whichfacilitates DNA repair and minimises accumulation of replication-mediated mutation.
Alternatively, damaged stem cells can be eliminated by apoptosis and progression to moredifferentiated compartments of a tissue such as progenitor and functional compartments.
Competition of stem cells for occupancy of the tissue stem cell niche is likely to help in
eliminating damaged stem cells.
2.3.2. Radiosensitivity of tissue stem cells(29) The DNA repair capacity of tissue stem cells is reflected in their radiosensitivity.
Radiosensitivity of stem cells can be assessed by clonogenic assays in vitro or in vivo usingtransplantation or in situ techniques. The results of such analyses are shown for epithelialcolony-forming cells (Fig. 2.3.). However, these assays generally cannot distinguishradiosensitivity of stem cells and progenitor cells of epithelial tissues since both types can beclonogenic cells whenever necessary.

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Fig. 2.3. Deduced survival curves for epithelial clonogenic cells in various tissues (Potten and Hendry,1983). (Permission needed)
J1, J2 – cells regenerating jejunal crypts.
G1, G2 – cells regenerating gastric crypts.
C – cells regenerating colonic crypts.
E1, E2, E3 – cells generating macroscopic epidermal clones.
S1 – cells regenerating spermatogenic tubules.
M – mammary tissue-forming units. Cells transplanted at 24 hours after irradiation in situ.
T – thyroid follicle-forming units. Cells transplanted at 24 hours after irradiation in situ.

(30) A moderate PLDR capacity was demonstrated for clonogenic cells of rat mammary
gland, thyroid, liver and bone marrow (Gould et al., 1984; Mulcahy et al., 1980; Jirtle et al.,
1985; Kamiya et al., 1991). A general feature in the epithelial systems was the increase insurvival when the cells were left in situ for 24 hours before transplantation in vivo to measurecolony formation. This had the effect of shifting the survival curve to higher doses (Fig. 2.4.).
The doses used in those PLDR experiments were mostly >6 Gy, and hence any changes in the‘α’ component at lower doses were not directly measured. But the survival curve, assessed
after a 24-hour recovery period, did have more curvature, i.e. the α/β ratio was lower, whichindicates that the decrease in α was more than any decrease in β. Examination of the effect offurther delay times before assay was performed using quiescent liver in vivo, with
hepatocytes transplanted into fat pads for colony growth (Fisher et al., 1988). For theseparticular cells, the change at 24 hours was a decrease in survival-curve slope, which
continued decreasing to the maximum 11 months delay examined. Cell proliferation was
insufficient to explain the long-term reduction in radiosensitivity in terms of a dosedependent replacement of damaged cells. Although there was a reduction in the frequency ofcells with micronuclei postirradiation, the magnitude of this decrease was relatively small.
Thus the long-term increase in clonogenicity could be explained only partially in terms oflong-term repair of chromosome injury, assessed by the production of micronuclei. In
addition, dose fractionation experiments were conducted, where the hepatocytes were assayed
for survival either early or late after irradiation in situ (Fisher and Hendry, 1988). When the34

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assay was delayed 10 months, the value of α showed a tendency to decrease only slightly.
The β component showed the greatest decrease with time, and the α/β ratio (1-1.6 Gy at 24hours) remained low but slightly increased to 1.9-2.1 Gy at 10 months. It should be noted thatthese tissue systems (thyroid, mammary gland, liver), all have different kinetic, lineage, andstructural properties, and hence postirradiation temporal changes may be tissue specific.
These clonogenic assays also showed that bone-marrow-derived progenitor cells were moreradiosensitive than epithelial clonogenic cells (Fig. 2.4.) (Hendry, 1985). The exact molecularmechanism of PLDR has not been elucidated, but ATM, the master gene of radiation damage
response, has been suggested to be involved in the process although a number of uncertainties
are yet to be resolved (Lobrich and Jeggo, 2005).

Fig. 2.4. Survival curves assessed in vivo by transplantation. Dashed lines: cells transplanted
immediately after irradiation. Solid lines: cells transplanted at 24 hours after irradiation (or 3-4 hours
in the case of marrow Pre-GM-CFS). The separation between the dashed and solid lines for eachtissue type indicates PLDR; note the positive effect in the case of mammary and thyroid epithelial
cells, and liver (hepatocytes), and in contrast the negative effect for marrow CFU-S considered due toa radiation-induced differentiation effect (Hendry, 1985). (Permission needed)(31) Recent technological advances enabled in vitro propagation of relatively pure
population of tissue stem cells which permits direct analyses of tissue stem cells. Humanbone-marrow-derived clonally-expanded MSCs were directly analysed for theirradiosensitivity. It was found that they were more radioresistant than human lung and breast
cancer cell lines and this was found to be due to a better antioxidant capacity of the cells(Chen et al., 2006). As discussed in Annex F, skin stem cells when tested in vitro were moreradioresistant than progenitor cells (Harfouche et al., 2010).
(32) A summary of radiosensitivities in relation to cellular stage in various hierarchicallineages is given in Table 2.1. Of note is the high radiosensitivity observed for cells that35

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undergo predominantly an apoptotic form of cell killing, e.g. some intestinal stem cells(ISCs), and types A, intermediate, and B spermatogonia.
Table 2.1. Radiosensitivity in relation to hierarchical status (updated from Potten and Hendry, 1983).
Tissue hierarchy
Radiosensitivity
Annex
D0 (Gy)Bone marrow, stem/progenitor(clonogenic)
Transit granulocytic
Transit erythroid
Mature cells
Fibroblastoid CFU-F

High
MediumVery highLow
Medium0.8-1.2
1.2-1.8
0.5-0.7
2.2

Medium~2

Thyroid stem cells

Medium2.0-3.5

C

Intestinal epithelium
some P4 stem cells (SI)CBCC
m-TertClonogenic cells (SI and colon)
Transit cells
Functional cells

Very highMediumResistantMediumLow
Very low

0.1-0.2
1.0-

D

Epidermis: stem (clonogenic) cells
Transit cells
Functional cells

MediumLow
Very low

1.4-

F

Testis Spermatogonia stem(clonogenic) cells
Type A
Intermediate
Type B
Functional (spermatocytes)
Sertoli cells

MediumHigh
Very highVery highLow
Very low

1.7-2.4
0.3-0.8
0.3
0.2
-

-

Mammary stem cells

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A

B

CFU-F, fibroblastoid colony-forming units; SI, small intestine; CBCC, crypt base columnar
cells.
2.3.3. Quiescence/dormancy of stem cells and DNA damage response(33) Mouse embryos before the compaction stage were shown to possess a unique p53dependent S-phase checkpoint with no activation of p21 (Shimura et al., 2002; Adiga et al.,2007). ES cells derived from the blastocyst stage were shown to lack p53-dependent p21activation after X-irradiation (Aladjem et al., 1998; Malashicheva et al., 2000). The cells ofthese stages are rapidly dividing, yet their DNA damage response differs considerably fromthat studied in cells in vitro.
(34) The DNA damage responses of adult tissue stem cells also vary, especially whenanalysed in the context of tissue microenvironments. For example, the P4 stem cells of mousesmall intestine are known for their high sensitivity to radiation induced apoptosis (Potten,
1977; Potten et al., 2002; Potten, 2004b) (Annex D). This altruistic cell death was thought to36

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be a mechanism to eliminate damaged cells and therefore to maintain genomic integrity.
Interestingly, some of the P4 cells were the first to undergo DNA replication after irradiationand pass through the p53/p21 repair pathway (Potten et al., 2009). Apoptosis of P4 stem cells
occurs in two phases. Early p53-dependent apoptosis at 4.5 hours after irradiation was
induced at doses below 1 Gy and delayed p53-independent apoptosis at 24 hours after higher
doses such as 8 Gy (Dove et al., 1998). Absence of the early p53-dependent apoptosis of
crypt stem cells in p53-null mice was restored when combined with the homozygous loss of
the DNA-PKcs gene (Gurley et al., 2009).
(35) In addition to P4 stem cells, Lgr5+ crypt base columnar cells (CBCCs) were claimed
to be the primary stem cells. These cells divide rapidly at a cycling time of 24 hours and areless sensitive to apoptosis (Barker et al., 2007). Extensive evaluation of the past and more
recent publications, however, led to a conclusion that the P4 cells are the likely stem cells ofsmall intestine, and that the CBCCs are the intermediate progenitors possibly differentiating
into Paneth cells (Potten et al., 2009). Furthermore, a very small subpopulation of cells was
identified among the P4 stem cells of mTert-expressing mouse intestinal crypt. These mTert+
cells were quiescent and did not exhibit apoptotic cell death even after 10 Gy (Montgomery
et al., 2011). They gave rise to all the cell types in the small intestine, including Lgr5+ cells.
Although further experiments need to be conducted, mTert+ P4 cells are probably the mostprimitive stem cells of the small intestinal mucosa. They are rare and quiescent, and they areinsensitive to radiation-induced apoptosis. This suggests that sensitivity to altruistic cell deathmay not be a universal radiation response of tissue stem cells.
(36) Dormancy or quiescence is a general feature of many tissue stem cells. For thequiescence of stem cells, the damage sensor ataxia telangiectasia mutated (ATM) serves anessential role for HSCs (Ito, 2004). ATM-/- mice displayed premature depletion of HSCs in
the bone marrow. The ROS level was high in ATM-/- mice and the high ROS level activated
p38 mitogen-activated protein kinase (MAPK) to force the quiescent HSC into cell cycling,which then resulted in the exhaustion of HSCs (Ito et al., 2006; Liu and Finkel, 2006). Thus,
cellular senescence eliminates overly-replicated stem cells, and quiescence is a mechanism tomaintain stem cell potential. Further studies demonstrated that p53 and p21 are also involvedin the quiescent state of HSCs (Liu et al., 2010; Cheng et al., 2000).
(37) The DNA damage response of stem cells includes loss of stemness (characteristics
which underlie self-renewal and the ability to generate differentiated progeny) which resultsin differentiation. A recent study has demonstrated that melanocyte stem cells undergo
terminal differentiation in the niche when exposed to radiation. ATM was found to be
involved in this terminal differentiation of melanocyte stem cells since the lack of its function
sensitises mouse skin to radiation induction of hair greying (Inomata et al., 2009). As in thecase of quiescence, p53 is also involved in regulating cellular senescence, in addition to ATM
kinase (Vigneron and Vousden, 2010).
2.3.4. DNA repair in stem cells(38) DNA repair in tissue stem cells might well differ from cells of other types since it is
known that the DNA repair pathway is dependent on their stage of differentiation. For
example, the nucleotide excision repair pathway was reported to be attenuated in terminallydifferentiated cells (Rasko et al., 1993; Nouspikel and Hanawalt, 2002; Hsu et al., 2007).
Mouse models defective in excision repair pathways exhibited tissue specific differences inmutagenesis and carcinogenesis, demonstrating that the repair system may differ between
stem cells of different origins (Wijnhoven et al., 2007). It is tempting to speculate that the
DNA repair pathways are responding to the demands of specific cell types. Mice have been
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created with specific defects in damage response and repair, and they provide excellent
models to study the role of tissue stem cells. As for the ageing of mice, the effect of repair
defects usually manifests more severely in tissues of rapid turnover such as haematopoietic
tissue (Park et al., 2005). HSCs from ageing mice were shown to have upregulated expressionof a series of stress-responsive genes, suggesting a strong correlation of ageing, DNA damage
and stress responses (Chambers et al., 2007). These studies demonstrate that DNA repair anddamage response play important roles for tissue stem cells, to stay quiescent and to preserve
genomic stability.
(39) Ionising radiations induce DSBs which are repaired either by HR or NHEJ. HR ispotentially error-free since it takes place in S and G2 phase cells to repair the damaged regionof DNA by copying the intact counterpart of the sister DNA strand. SLDR was shown to be
dependent on Rad54, and therefore, represents the repair activity of HR (Rao et al., 2007).
NHEJ takes place in non-cycling cells and in all cycle phases to a varying degree, and isdependent on the repair proteins Ku70, Ku80 and DNA-PKcs. PLDR is the repair of noncycling cells and thus represents the repair activity of NHEJ.
(40) ES cells with either Rad54-/- or Ku70-/- were found to be equally sensitive toionising radiation, showing the importance of both repair pathways (Gu et al., 1997). In
contrast, although adult Ku80-/- mice and DNA-PKcs-/- mice are sensitive to radiation, adult
Rad54-/- mice exhibit hypersensitivity only when combined with the DNA-PK deficiency(Essers et al., 2000). A series of mice defective in DSB repair has been generated andcharacterised (Brugmans et al., 2007). Among these mice, DNA ligase IV defective mice
exhibit premature ageing of HSCs (Nijnik et al., 2007). These indicate that NHEJ is likely tobe the major pathway of radiation damage repair in adult tissue stem cells. The
dormancy/quiescence of tissue stem cells is essential especially for the tissues producing vast
numbers of cells such as haematopoietic and gastrointestinal (GI) tissues. In the non-cycling
quiescent tissue stem cells, the NHEJ pathway is the only way to repair DNA damage. Thus,
the NHEJ pathway is associated with PLDR, which is defined operationally as the repair
occurring in the stationary phase non-cycling cells. Hence it is reasonable that tissue stem
cells exhibit a large capacity of PLDR as shown by in vivo and in situ clonogenic assays (Fig.
2.4.) (Hendry, 1985).
(41) Quiescence of stem cells poses two problems to the strategy of tissue stem cells inmaintaining the integrity of the genome. Firstly, quiescent stem cells rely on NHEJ, but thisrepair pathway is considered to be error-prone. In addition, DNA damage accumulates in thequiescent stem cells, as demonstrated in HSCs by the occurrence of spontaneous γH2AX fociin ageing mice (Rossi et al., 2007). Radiosensitivity was tested for haematopoietic stem andprogenitor cells (HSPCs: Sca-1+, CD34-), common myeloid progenitors (CMPs: Sca-1-,
CD34+) and granulocyte/macrophage progenitors (GMPs: Sca-1-, CD34+). Theirradiosensitivity as tested directly by the in vitro clonogenic assay demonstrated that quiescent
HSPCs were more radioresistant than CMPs and GMPs, as expected (Mohrin et al., 2010).
However, the frequency of chromosome aberrations after 2 Gy irradiation, as measured by
SKY analyses, is higher by more than two-fold for the quiescent CD34- HSPCs than the
CD34+ two cell types. A stem-cell-enriched haematopoietic cell population was reported to
exhibit similar chromosome sensitivity to that of peripheral lymphocytes, but the cell
population in that study was CD34+. Therefore, conclusive judgment cannot be made atpresent from the study on the chromosome sensitivity of CD34+ HSPCs (Becker et al., 2009).
(42) These results may suggest that PLDR executed by the NHEJ pathway confers better
survival of quiescent tissue stem cells, but it may also cause more chromosome mutations.
Consistent with this notion, the classic study of PLDR using V79 cells also indicated that theirradiated cells kept in the stationary phase exhibited better survival, but the HPRT mutation
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frequency stayed the same irrespective of the holding time (Thacker and Stretch, 1983). Yet,a recent study indicated that irradiated human diploid fibroblasts have less chromosome
aberrations when they are in the non-cycling G0 phase than in cycling G1 phase (Liu et al.,2010). Further analyses need to be made to clarify the role of DNA repair in relation tocolony survival and mutagenesis of quiescent tissue stem cells.

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2.4. Ageing and exhaustion of tissue stem cells

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2.3.5. Cairns’ hypothesis: suppression of replication-mediated mutation in tissue stem
cells(43) Since high dose radiation is not common in natural conditions, coping withradiation damage is not of importance to the normal maintenance and function of tissue stem
cells. Rather, avoiding naturally occurring mutagenic events is more important. Among such
events, DNA replication is an unavoidable source of mutation. An interesting hypothesis was
proposed by Cairns (1975). The ‘immortal strand’ hypothesis proposed asymmetricsegregation of DNA strands to minimise the replication error in the tissue stem cells. The
stem cell retains the template DNA strand after a round of DNA synthesis, while theprogenitor cells inherit the daughter strand. Since the template strand is never replaced, thereplication error is minimised for tissue stem cells, while the strands with possible errors are
passed to the progenitors which are eventually lost by differentiation/maturation intofunctional cells. The evidence supporting the hypothesis was presented in mice injected with3
H-thymidine at infancy which carried the long-term LRCs in the stem cell region of rapidly
proliferating intestinal crypts (Potten et al., 2002). In order for the template strand to stay on
one side of the sister chromatids, there should be no recombination in the tissue stem cells.
This requirement is likely to make stem cells to be ‘recombination minus’ which then leaves
NHEJ as the only legitimate repair system in stem cells. Also, the template-strand hypothesis
requires that all sister chromatids with the template strands have to segregate to the stem-cellside of the spindle poles at mitosis, with the one-to-one connection of old centrosomes andold centromeres. However, such requirements still have to be shown on a molecular basis.
(44) Proponents of the immortal strand hypothesis rely mostly on histological data
indicating the presence of LRCs, and P4 stem cells in mouse intestinal crypts were shown toretain 3H-thymidine or bromodeoxyuridine (BrdU) for a long time (Potten et al., 2009).
Asymmetric chromosome segregation is currently not generalised for all tissue stem cells,
and the immortal strand hypothesis remains under critical debate (Rando, 2007; Lansdorp,2007). It was demonstrated that asymmetric segregation of DNA strands does not take placeat least in HSCs and in hair follicle stem cells (Kiel et al., 2007; Waghmare et al., 2008).
However, strand-specific segregation was reported on chromosome 7 in mouse neuronal cells(Armakolas and Klar, 2006). Furthermore, CO-FISH technique was used to address the
problem. The results indicated that whereas the segregation of sister chromatids was random
in mouse fibroblasts and ES cells, the segregation in mouse colon cells was not random(Falconer et al., 2010). The asymmetric segregation of the immortal sister chromatid requires
suppression of the HR pathway, and at least, this is consistent with the reliance of tissue stem
cells on the NHEJ repair pathway, rather than the HR pathway. The immortal strand
hypothesis is likely to remain controversial until a more definitive analysis becomes possible.

2.4.1. Mortal nature of tissue stem cells

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(45) Tissue stem cells divide and replenish the tissue for the entire life of an individual.
Tissue stem cells were once thought to be immortal and immune to cellular senescence. Oneof the determinants of cellular senescence is the loss of telomeres through DNA replication.
Rapidly replicating cells therefore usually have telomerase activity to avoid telomere
shortening (Blasco, 2007). Indeed, tissue stem cells were reported to possess telomerase
activity, and this has been confirmed in a variety of tissue stem cells (Harrington, 2004).
However, mouse and human HSCs nevertheless lose telomeric DNA with serial passages ofcells in vitro and in vivo ageing of animals (Vaziri et al., 1994; Allsopp et al., 2001). Thus,
adult tissue stem cells are unlikely to be immortal, although their division potential isenormous, e.g. as shown in bone marrow and intestine. Telomerase-deficient mice werereported to exhibit pronounced ageing with atrophy in rapidly proliferating tissues such asbone marrow, intestine and testis (Lee et al., 1998; Rudolph et al., 1999). There is now ample
evidence to demonstrate that telomere shortening takes place in stem cells of an ageing body
(Flores et al., 2006).
2.4.2. Telomere length of stem cells(46) As discussed, tissue stem cells have limited telomerase activity so that erosion of
telomere ends through rounds of DNA replication is inevitable. Therefore tissue stem cells
have to possess mechanisms to prevent the loss of telomeres. One way is to have efficient
repair of DNA damage, and failure to do this results in the loss of cells which necessitatescompensatory replication of stem cells in a tissue. Therefore, damage checkpoints and DNA
repair are important for tissue stem cells. Also, quiescence in the well-protected
microenvironment of the tissue stem cell niche acts to promote the genomic integrity of tissue
stem cells. Indeed, tissue stem cells in their niche seem to have the longest telomeres, as
quantitative FISH has revealed for mouse hair follicles, small intestine, testis, cornea andbrain (Flores et al., 2008). It is interesting to note that in this particular study, fluorescentsignals for telomeres of the Lgr5+ CBCCs were less than those of the P4 cells, implying theformer being higher in the hierarchy of ISCs. Thus, even though tissue stem cells havetelomerase activity and the longest telomeres, they nevertheless shorten their length in ageing
mice. Shortening of telomeres can also be accelerated when HSCs are forced to replicate byserial bone marrow transplantation (Allsopp et al., 2001).
2.4.3. Telomere shortening and carcinogenesis(47) When telomeres become critically short, the chromosome ends lose protection. The
exposed chromosome ends are recognised as DSBs. DSBs are frequently rejoined incorrectly
to create dicentric chromosomes which either block cell division or break apart to create new
double-strand ends at mitosis. This breakage-fusion-bridge cycle can induce genomic
instability in the cells, as human embryonic kidney cells undergoing replicative shortening oftelomeres were shown to exhibit chromosome instability (Counter et al., 1992). Telomere
shortening and chromosome instability lead to other consequences by activating DNA
damage responses including focus formation of γH2AX, p53-binding protein 1 (53BP1),
meiotic recombination 11 (Mre11) complexes and phosphorylated ATM (Takai et al., 2003).
Resulting DNA damage responses culminate in activation of a series of p53-mediatedresponses including apoptotic cell death and/or cellular senescence (Karlseder et al., 1999;
d'Adda di Fagagna et al., 2003). Senescence acts as a powerful block for carcinogenesis, as
telomerase-deficient mice were found to resist chemical induction of skin cancer (GonzalezSuarez et al., 2000). Cellular senescence and apoptosis led to loss of stem cells, forcing them
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to undergo further replication in order to maintain tissue homeostasis. This creates a vicious
cycle of the additional loss of stem cells in tissues. Erosion of telomeres due to natural orforced replications thus leads to stem cell exhaustion, which is a hallmark of ageing tissues.
(48) Cellular senescence brought about by short telomeres may act as a block to
carcinogenesis. However, short telomeres and the resulting genomic instability are associatedwith cancer induction in mice and humans (Murnane, 2012). Although mice lacking
telomerase exhibit premature ageing phenotypes, the same mice on the p53 null genetic
background are highly prone to developing epithelial cancers (Artandi and DePinho, 2010).
Interestingly, the p53-null allele does not have to be homozygous, and heterozygosity wasenough for high rates of cancers in these mice. Also, a p53+/- genetic background restituted
the premature ageing and stem-cell exhaustion phenotypes of telomerase- mice (Flores et al.,2009). Thus, the status of p53 is the major determinant of the two opposing outcomes oftelomere erosion in tissue stem cells: namely, loss of cellularity in ageing tissue, andunregulated cell proliferation in carcinogenesis.

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2.5. Tissue stem cell niche

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2.5.1. Stem cell niche(49) In homeostasis conditions, a tissue stem cell in adults is considered to divide
asymmetrically to produce a stem cell and a progenitor cell. This asymmetry of cell division
requires spatial asymmetry of the stem cell microenvironment, and the stem cell niche
provides such a cue for the asymmetry (Watt et al., 2000). In the stem cell niche, stem cells
attach proximal to specific stromas while the daughter cell positions are distal to them.
Asymmetric expression of adhesion molecules on one side of a cell assures the specificinteraction, which in turn results in the signalling by short acting factors mainly tissue
specific cytokines to maintain the stemness of the cells. In addition, a study on the germline
stem cells in the Drosophila testis demonstrated that an alignment of centrosomes plays acritical role in the division of stem cells perpendicular to the stroma (Yamashita et al., 2007).
In the Drosophila testis, the stem cells are positioned proximal to the stromal cells in the
germline niche and green fluorescent protein (GFP)-tagged centrosomes align with theproximal to distal direction. This structural feature of stem cells and their centrosomes areshared by mammalian tissue (Fuchs, 2004).
(50) Positional information of the stem cell niche is therefore pivotal to the hierarchial
structuring of cells in a tissue by activating expression of a series of genes. This means thatthe gene expression patterns and the fates of stem cells, progenitors and terminallydifferentiated cells could be reversibly modified by changing the positional information, orby directly modifying the expression of fate-determining genes. In the Drosophila testis,progenitor cells were demonstrated to become stem cells by taking over the vacant stem cellniche (Cheng, 2008; Cheng et al., 2008). This dedifferentiation is associated with a specificpattern of gene expression. Direct manipulation of genes led to dedifferentiation and transdifferentiation of cells as demonstrated by a case of Pax5-deficient mature B cells which
dedifferentiate and then trans-differentiate into T cells (Cobaleda et al., 2007). An extreme
case of dedifferentiation and trans-differentiation is the conversion of mouse fibroblasts to
induced pluripotent stem (iPS) cells by ectopic expression of 4 stem-specific genes: octamerbinding transcription factors 3/4 (Oct3/4), sex determining region Y-box 2 (Sox2), c-Myc andKruppel-like factor 4 (Klf4) (Takahashi and Yamanaka, 2006). All of the recent studies thus
indicate that the fate of cells is re-programmable.

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2.5.2. Stem cell niche as a shelter(51) The stem cell niche was shown to promote rapid establishment of the stem cell pool
after genotoxic insults such as radiation exposure, and transplantation of MSCc rescues
lethally-irradiated mice through their HSC-niche modulating activity (Lange et al., 2011). In
the steady state, the stem cell niche was shown to provide a shelter from various genotoxic
stresses. Tissue stem cells have to sustain themselves for the entire life span of an individual,
and they have a number of strategies to circumvent genotoxic stresses. One of the strategies
to escape from ROS is to rely on the intracellular antioxidants, as in the case of MSCs (Chenet al., 2006). Another strategy is to stay quiescent in a low oxygen environment, and the stem
cell niche of HSC is such an example (Suda et al., 2007).
(52) There are three niches of HSCs in the bone marrow, an osteoblastic niche, avascular niche and a medullary niche (Annex A) (Shiozawa and Taichman, 2012). HSCs in
the former niche are in close association with osteoblastic cells, thus two most important
targets of radiation carcinogenesis are sharing the same microenvironment in a tissue (Zhang
et al., 2003; Calvi et al., 2003). HSCs are quiescent and this state of cells is dependent on
their residence in the niche. The Tie2/angiopoietin 1 (Ang-1) signalling between the two cell
types regulate quiescence of HSCs in the osteoblastic niche (Arai et al., 2004). Quiescence isa property shared by stem cells in other tissues, including EpiSCs (Nishikawa and Ozawa,
2007). Also, many types of tissue stem cells, such as those of neural, mammary,mesenchymal and adipose tissues, as well as ES, favour a hypoxic condition for their stable
persistence in vitro and in vivo (Ezashi et al., 2005; Danet et al., 2003; Ivanovic et al., 2000;
Grayson et al., 2007; Zhu et al., 2005; Lin et al., 2006). The osteoblastic niche of HSCs ispoorly vascularised, and some HSCs were shown to be stained strongly by hypoxia-sensitive
pimonidazole, suggesting that the oxygen concentration of the niche is less than 2% (Parmar
et al., 2007). HSCs residing in the osteoblastic niche are those of primitive and lesscommitted types, and hypoxia is favoured to protect cells from endogenous ROS. Hence therole of protection given by the stem cell niche has to be considered when assessing the effectof radiation on tissue stem cells.
(53) Although hypoxia in the niche microenvironment is important, it is interesting tonote that an appropriate level of ROS is also required for the maintenance of genomic
stability in ES cells in culture (Li and Marban, 2010). The frequency of chromosome
aberrations analysed in ES cells decreased with decreasing levels of oxygen, but increased
when the cells were treated with antioxidants. This increase was associated with the depletionof expression of repair-related genes when ROS were completely absent.
2.5.3. Stem cell competition for residence of the niche(54) In contrast to the classic concept of asymmetric cell division of stem cells, a numberof studies revealed that stem cells often divide symmetrically to produce two stem cells, or to
produce two committed stem cells/progenitor cells (Fig. 2.5.). In the former case, the surplusof stem cells can compete for residence in the niche, and inferior stem cells are eliminated. In
the latter case, a vacant stem cell niche is created with two committed cells leaving the niche.
These processes lead to turnover of stem cells, which once were thought to reside in a tissue
for a lifelong period.

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Fig. 2.5. Three division patterns of tissue stem cells.

(55) The first evidence of such competition and turnover comes from the analyses ofmutant stem cells of intestinal crypts (Potten et al., 2009). For example, a mutant stem celltakes over the entire crypt in 5 to 7 weeks in colon and 12 weeks in small intestine after
mutagen treatment of mice (Loeffler et al., 1993). Population expansion from a single stem
cell and the resulting monoclonality of the crypt have also been shown by the lineage tagging
of Lgr5+ stem cells in intestinal crypts. From the pattern of the clone size distribution, it was
concluded that ISCs are equipotential in replacing the neighbour, or being replaced by theneighbour, in a neutral drift fashion without directional pressures. Thus, ISCs are sometime
lost, or take over the entire crypt (Lopez-Garcia et al., 2010). The lineage tagging method
was applied to a variety of tissues and a similar competition and resulting turnover of stem
cells were demonstrated in testicular germline stem cells (Lopez-Garcia et al., 2010; Klein etal., 2010) and in skin stem cells (Clayton et al., 2007).
2.5.4. Competition of stem cells during establishment of the adult stem cell niche(56) Although dependent on the tissue type, the adult stem cell niche is usually
established around the postnatal period after birth. For example, the intestinal tract in themouse is formed as a simple tube of epithelial cells with high proliferative activity. The firstdifferentiation of these equipotential epithelial cells, or fetal stem cells, is the formation of thevilli at embryonic day 15, which are necessary for absorption of nutrition after birth. The
formation of crypts which provide an adult-type stem-cell niche for the maintenance of tissue
proliferation appears only at postnatal day 7 (Crosnier et al., 2006). It is important to note thatwhile the stem cells left aside in the niche of newly-formed adult-type intestine are few, thenumber of fetal stem cells in the fetal intestine is substantial. This suggests a strong
competition of stem cells for the occupancy of the niche. It is also interesting to note that theneonatal crypt is occupied by a polyclonal population of stem cells, and that the
monoclonality is established at about 2 weeks after birth (Schmidt et al., 1988). This
monoclonal conversion supports competition of fetal stem cells to result in the occupancy ofa niche by a dominant clone.
(57) Haematopoietic development is first detected in the yolk sac region of embryos,
which then shifts to an aorta/gonad/mesonephros site. A major site of haematopoiesis at the
fetal stage is the liver, while the site in the adult is the red bone marrow. HSCs in the fetalliver migrate and settle in the bone marrow niche (Orkin and Zon, 2008). Although the
migration of HSCs from liver to bone marrow is generally preserved among mammalian
species, there appears to be distinct differences between the temporal patterns and major sitesof haematopoiesis in the developing fetus of mice and humans: e.g. in the developing human43DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
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fetus, marrow steadily increases haematopoietic activity in the second half of gestation. In
contrast, in the mouse, the liver is the dominant haematopoietic site from mid-gestation tobirth, and only very late in gestation and into early postnatal period is there active migrationof HSCs from the liver to skeletal sites. The process of ‘adult niche development’ appears tooccur at different times postnatally in the two species.
(58) The fetal liver HSCs differ in their characteristics from adult bone marrow HSCs, inthat the former are rapidly cycling while the latter are in a state of quiescence. The gradualshift in characteristics of HSCs takes place in the mouse 3 weeks after birth. Redistribution tobone marrow is selective rather than random at least in the mouse, so that HSCs are deficientin engraftment when they are transiting S/G2/M while those in G1 successfully settle in thebone marrow niche (Bowie et al., 2006). Thus, the homing into the adult stem-cell niche
during the neonatal stage development functions as a selective process in the case of mouse
HSCs, where stem cells compete for residence the niche with retention of favourable cells
and elimination of unwanted cells (Fig. 2.6.).

Fig. 2.6. Stem cell competition in the neonate. The competition is particularly strong when the adulttissue stem cell niche is established during the neonatal stage. (permission needed)

2.5.5. Effects of radiation on stem cell competition(59) Irradiation affects the competition of HSCs for their residence in the bone marrowniche. When two marked populations were mixed and transplanted to lethally-irradiated mice,
both populations contributed to the reconstituted HSCs equally. However, when one of thetwo populations was first exposed to 1 Gy and subsequently mixed with the second nonirradiated population prior to transplantation, the non-irradiated population predominated in
the reconstituted marrow HSCs (Bondar and Medzhitov, 2010). In this competition, p53
played a crucial role in sensing the stress of irradiation, since the HSCs of a p53+/- or p53-/genotype exposed to radiation were not outcompeted by the unirradiated wild-type population.
The competitiveness was solely dependent on the p53 level, since bone marrow cells of
Mdm2+/- mice with higher levels of p53 protein were outcompeted by those from the wildtype mice even without irradiation. Somewhat similar observations were made using bonemarrow cells with the retrovirally-transduced p53 with functionally blocked dimerisation

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domain, in which the cells were dominant over the wild-type cells after 2.5 Gy X irradiation(Marusyk et al., 2010). Altogether, stem cell competition for the residence of the tissue stem
cell niche is sensitive to radiation stress which is sensed by p53.

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3. THE ROLE OF TISSUE STEM CELLS IN RADIATION CARCINOGENESIS

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3.1. The role of stem cells in radiation carcinogenesis

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(60) This chapter aims to depict subjects of importance for basic processes ofradiological health risk assessments and the development of guidelines deemed essential to
the ICRP system of radiological protection. The first two sections deal with the mechanismsof carcinogenesis in tissues with a special emphasis on the role of tissue stem cells and otherpossible target cells with regard to the LNT model and the RR model. The discussion thenmoves into the radiation biology of stem cells, and the dynamic nature of stem cellcompetition in the tissue stem cell niche. This tissue level dynamism is likely to play a role
when considering the dose rate effect, which in the past has been considered only from thecellular repair point of view. Stem cell competition is a new concept but it can explain somefeatures of radiation risk, and more importantly, the age dependence of radiation
carcinogenesis. Altogether, this chapter is devoted to bridge the advancement of knowledgeof stem cell biology and issues of importance to radiation risk assessment.
3.1.1. Multistage carcinogenesis(61) Cancer in adulthood is envisaged to arise as a result of accumulation of oncogenic
mutations occurring mainly after birth, whereas some childhood cancers are characterised bymutations acquired during fetal development or inherited from the parents. The incidence ofcancer in adults, especially solid cancer, exhibits a steady increase by age. Armitage and Doll
(1954) noted that this increase follows roughly the fifth power of age and proposed themultistage carcinogenesis model. This was later supported by the molecular analysis ofhuman colon cancer, in which the conversion of normal epithelial cells to adenoma and its
progression to carcinoma were shown to be associated with a stepwise acquisition of
mutations of oncogenes and tumour suppressor genes (Fig. D.8. of Annex D; Vogelstein et al.,1988). Acquisition of multiple mutations by spontaneous processes takes a long time, which
explains why adulthood cancers arise late in life.
(62) Cancers in childhood form a unique group of neoplasias, which occur before
puberty, between birth and 15 years of age. In contrast to adulthood cancer, childhood cancer
such as retinoblastoma was found to require only two steps (Knudson, 1971). This two-stepcarcinogenesis process can explain why childhood cancer occurs with relatively short latency
in early life before puberty. The reason for the difference in the number of steps (ormutations) for cancer of adulthood and childhood onsets is not fully understood. However, it
is likely that these two differ in the target cell types, with some of the former being fetal-stage
primitive cells and the resulting cancer often carries the suffix of “blastoma”, while the latterbeing adult tissue stem cells and progenitor cells.
(63) Animal experiments have shown that the process of stepwise carcinogenesis can be
categorised into four steps: the initiation step with the irreversible change of a normal cell
into a preneoplastic state, the promotion step with the proliferation and the clonal expansionof initiated cells, the malignant conversion step with an acquisition of neoplastic
characteristics of cells, and the progression step with further accumulation of changes in cellswith invasion to normal tissue territories. Each step is associated with functional changes ofgenes regulating cell proliferation, quiescence, differentiation, senescence and apoptosis(Perez-Losada and Balmain, 2003). These changes in gene functions are often brought about46

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by the genetic mechanism of mutation induction and by the epigenetic mechanism oftranscription factors, chromatin modifications, DNA methylation and regulatory microRNA(miRNA) (Sharma et al., 2007). However, the present report attempts to restrict thediscussion so as to focus on the multistage carcinogenesis model where mutations are thedeterminant. This restriction is made in order to simplify the discussion on the numericalaspects of radiation carcinogenesis. For the same reason, mechanistic models involving
epigenetics and promotion/progression aspects are not considered.
3.1.2. Target cells for carcinogenesis(64) The target cells for carcinogenesis are considered to be the tissue stem cells andtheir proximal progenitors (Reya et al., 2001). This assumption is considered reasonablebecause many characteristics of tissue stem cells resemble those of cancer cells, and theresemblance became particularly strong after the discovery of cancer stem cells in humanleukaemia (Lapidot et al., 1994). Cancer stem cells are also found in a variety of solid tumour
types arising in breast (Dick, 2003), brain (Singh et al., 2003; Hemmati et al., 2003), prostate(Lawson and Witte, 2007), liver (Roskams, 2006), and many other tissues. Cancer stem cells
resemble normal tissue stem cells in many respects. They have capacities to self-renew and
can initiate tumour formation when transplanted into appropriate hosts. Cancer stem cells
exhibit the SP phenotype and therefore are resistant to chemotherapeutic agents as are normal
tissue stem cells. Also, cancer stem cells in vivo are often quiescent, in contrast to rapidly
proliferating non-stem cancer cells because of constitutive upregulation of damage
checkpoints (Bao et al., 2006).
(65) These similarities suggest that cancer stem cells may arise from normal tissue stem
cells and those of their proximal progenitors, which are able to regain stemness. A stem cellorigin of cancer is suggested because the pool size of tissue stem cells in some cases
correlates with the risk of carcinogenesis. For example, the number of mammary stem cells(MaSCs) is determined by the level of hormones in utero, and this suggests influences onsubsequent breast cancer risk after birth (Trichopoulos, 1990). Similarly, body size of
newborn babies is known to correlate with the leukaemia risk (Caughey and Michels, 2009).
Involvement of insulin-like growth factor 1 (IGF1) was recently implicated in this correlationsuggesting that either a larger target cell number or a higher proliferation rate of HSCs isrelated to leukaemogenesis (Chokkalingam et al., 2012).
(66) A stem cell origin of cancer is also reasonable, because this is the only cell typewhich has a long enough time of residence in the body to accumulate multiple mutations togain malignant phenotypes, while progenitor cells do not have sufficient time (ICRP, 2005).
It is interesting to note that the small intestine with high proliferation is refractory to
carcinogenesis in humans, while the adjacent large intestine is prone to radiation
carcinogenesis, demonstrating that not all stem cells give rise to cancer. A high sensitivity toapoptosis of small intestinal P4 stem cells was proposed as a reason for an extremely low
cancer incidence in the small intestine (Li et al., 1992; Potten et al., 1992). Therefore, in
addition to the number of target cells, their behaviour also plays a decisive role incarcinogenesis.
(67) There are examples, which suggest that lower-ranking stem cells and progenitor
cells can be the target for carcinogenesis. The Cre recombinase-mediated loss of the APC
gene in Lgr5+ CBCCs, lower ranking to the P4 stem cells, was found to result in theformation of full adenomas (Barker et al., 2009). Also, in the human brain, precancerous
lesions are known to arise first in the differentiated compartment, and poorly differentiatedglioblastoma then evolves from a well-differentiated astrocytoma with a latency of 5-10 years
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(Klihues and Cavenee, 2000). At the molecular level, inactivation of Ink4a/Arf was shown totrigger dedifferentiation of astrocytes, and further introduction of constitutionally active EGF
receptor (EGFR) conferred the malignant glioma phenotype to the cells (Bachoo et al., 2002).
(68) In haematopoietic malignancy, progenitor cells retain a large proliferative capacity
and are a common target of carcinogenesis, as many leukaemias and lymphomas carrycommitted phenotypes of particular lineages. In addition, progenitor cells were shown tobecome leukaemia stem cells by direct introduction of a fusion oncogene, MLL-AF9(Krivtsov et al., 2006). The classic mouse model of radiation-induced thymic lymphomas wasshown to arise from the CD4-/CD8- progenitors in the thymic environment (Kominami andNiwa, 2006). Proliferation of leukaemia cells requires a bone marrow microenvironment, andthat of lymphoma cells needs a lymph node environment. These two haematopoietic
malignancies carry phenotypes of stem cells and committed progenitor cells, respectively.
(69) Skin is another tissue where progenitor cells in addition to the stem cells can form
cancer. As discussed in Annex F, three cancer types are known in the skin; BCC, SCC andpapilloma (section F.5, Fig. F.5.). EpiSCs were proposed to give rise to BCC commonly
found in people of the European ancestry, early progenitor cells to give rise to moremalignant SCC, and late progenitor cells to form benign papillomas. Although this is aninteresting and informative model, how the early progenitor cells can resist the polarised flowof cells without being discarded is elusive.
3.1.3. Role of radiation in carcinogenesis(70) The current model of radiation carcinogenesis assumes that radiation acts as amutagen, and gives possibly one or two carcinogenic mutations to a target cell (ICRP, 2007).
Radiation is known to induce DSBs especially those with clustered DNA damage, which areprone to inducing large mutations such as deletions and translocations. Induction oftranslocations follows an LQ dose response, while induction of small deletions is likely tofollow a linear dose response. Deletion mutations inactivate tumour suppressor genes, whiletranslocations activate proto-oncogenes by juxtaposing them to strong transcription promoterelements or making fusion genes with oncogenic functions. These targeted actions ofradiation are the theoretical foundation for the LNT model, which is used for projection ofhealth risk at low dose and low dose rate.
(71) The direct involvement of radiation in inducing the oncogenic mutation has beentested experimentally by examining the presence of the transcripts of the rearranged genes,characteristic of leukaemias and thyroid cancer, in cells in culture. However, the dose
required for such rearrangements was found to be 50 to 100 Gy, which is extremely highcompared with the real situation where a few Gy induced those cancers (Ito et al., 1993a; Itoet al., 1993b). In addition, the characteristic rearrangement of RET/PTC elements inchildhood thyroid cancer showed a strong age dependence of occurrence, casting doubt onwhether such translocation is really due to radiation exposures (Annex C). These indicate thata signature of radiation in radiation-induced cancer has yet to be identified.
(72) Recently, an interesting mechanism was discovered which suggests a direct
involvement of radiation in induction of multiple carcinogenic mutations. In contrast to the
stepwise acquisition of mutations, genomic analyses of human cancer have demonstrated that
2-3% of all cancers and about 25% of bone cancers acquire multiple mutations by a single
event (Stephens et al., 2011). Chromothripsis, as it is called, is multiple genomic
rearrangements with sharply circumscribed regions of one or a few chromosomes,
crisscrossing back and forth across involved regions. Involvement of a micronucleus in thegeneration of chromothripsis was demonstrated recently (Crasta et al., 2012). Micronuclei are48

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easily induced by radiation through a single hit process, yet its role has been implicated incell death, and not in carcinogenesis. Thus, the role of chromothripsis in radiation
carcinogenesis needs to be further investigated.
(73) In addition to these targeted actions, radiation is also known to act in a non-targetedfashion, which includes bystander effects and the induction of genomic instability especiallyin the low dose range (ICRP, 2007). Radiation has long been known to induce transientchanges in gene expression, but some of these changes persist for a long time after irradiation.
Indeed, the epigenetic mechanism of DNA methylation was recently shown to underlie thenon-targeted effect of radiation (Goetz et al., 2011). Nevertheless, involvement of nontargeted and epigenetic effects have not yet been fully shown, since the deviation of dose
responses for incidence and mortality from linearity is still weak in the low dose range forcancer among atomic bomb (A-bomb) survivors (ICRP, 2005, 2007). Furthermore, radiationis also known to change cell-to-cell and cell-to-tissue interactions in a tissue’s
microenvironment. In the case of the mammary gland, radiation has been shown to induce
TGFβ from the matrix, which plays a significant role in mammary carcinogenesis (Nguyen etal., 2011). Irradiation modifies stem-cell-to-niche interaction by giving selective advantage to
the Notch1 and p53-null stem cells for residence in the bone marrow niche, contributing to
the further development of leukaemia (Marusyk, 2009; Marusyk et al., 2010). These studies
suggest a variety of roles played by radiation in the induction of cancer. Nevertheless, the
LNT model based on the targeted mechanism of radiation is still used widely to assess therisk at low dose and low dose-rate exposures since it fits fairly well with the epidemiological
data of induction of cancer in radiation exposed human populations.
3.1.4. Models and the risk of radiation carcinogenesis(74) The radiation dose-incidence relationship regarding solid cancers is fairly linear for
A-bomb survivors, although there is recent evidence for curvilinearity when the dose range islimited to <2 Gy (Ozasa et al., 2012). In addition, a majority of solid tumours among thesurvivors occurred at a so-called ‘cancer-prone’ age, with a similar age-incidence trend as in
the non-exposed population, but with the incidence being higher among the former in a dosedependent manner. These effects are consistent with the assumption that radiation contributes
one mutation out of multiple mutations necessary for full malignancy (ICRP, 2007).
Assuming five mutations for cancer development, four of them are contributed by mutagenic
factors other than radiation, including errors in DNA replication and chromosome segregation,and mutations induced by internal/external mutagens. Under this scenario, the RR model,
also called the multiplicative model, is adequate for the risk assessment of cancer ofadulthood onset since radiation is expected to increase, linearly with the dose, the proportionof cells with five mutations in populations, which already gained four mutations or to gain
four mutations in the future. Thus, the net effect is that radiation increases the risk of cancer
linearly to the dose and proportionally above the background incidence. However, the above
considerations of mutations indicate that the role of radiation is relatively small incomparison to that of other mutagenic factors. This raises a question of whether or not it isreasonable to call a cancer radiation-induced, since radiation is not a major contributor tocarcinogenesis. One may call such cancer radiation-associated, or radiation-related. However,
the term radiation-induced has been kept for historical reasons and for simplicity of the
discussion. In contrast to the RR model, the AR model (sometimes called the additive model)
assumes that the radiation risk is independent of the background risk and depends linearly on
the dose. Today, both RR and AR models appear to fit well the risk of solid cancers in the A-

49

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bomb survivors’ cohort, with coherent results about the modifying effects of age at exposureand attained age (Ozasa et al., 2012).
(75) Apart from its role as a descriptive model, the RR model implies an interesting
practical application in the prospective management of carcinogenic risk of people after
exposure to radiation, which is assumed to be impossible because of the nature of radiation as
an absolute mutagen. As mentioned above, with the RR model, the extent of the risk from adose of radiation is proportional to the background incidence. Hence any action taken which
reduces the background incidence may also result in a reduction in the radiation-related
increase of EAR. An example of this can be seen in the case of lung cancer from residential
radon among smokers and non-smokers. The ERR for radon is similar at about 0.16 per 100
Bq/m3 for both smokers and non-smokers, yet the background incidence at age 75 years
differed about 25-fold between smokers and non-smokers (Darby et al., 2006). In
experimental systems, caloric restriction was shown to reduce the spontaneous occurrence of
myelocytic leukaemia in mice. In addition, the reduction was also noted in irradiated mice
when caloric restriction was applied even after the radiation exposure (Yoshida et al., 1997).
Application of caloric restriction and other measures were reviewed recently for their various
effects on the incidence of radiation-induced cancer in experimental animals (Oliai andYoung, 2013). These experimental findings imply that human health promoting actions likestopping smoking and improving dietary habits might lower not only the backgroundincidence but also the radiation-related increase of some types of cancer. The benefit of suchmeasures may be expected in theory for the cancer types where the RR model applies.
Epidemiological studies could be designed to test whether such measures effectively reduce
the future occurrence of cancer.
(76) In contrast to solid tumours, the dose response for leukaemia among A-bomb
survivors, especially acute myeloid leukaemia (AML), has a strong quadratic as well as alinear component (Hsu et al., 2013). In addition, AML has a relatively short latency time afterradiation exposure. These suggest the involvement of relatively fewer mutations such as two
or so for this neoplasm. If the number of mutations is small, radiation can act as absolute
carcinogen to supply all such mutations. Similarly, childhood cancer is known to be induced
by a relatively smaller number of mutations such as two with shorter latency than adult-onset
cancers, as discussed in section 3.1.1. The AR model is likely to fit with these cancer types,
leukaemia and childhood cancer, since radiation is sufficient to induce cancer regardless ofthe background incidence in a particular target population.
(77) From the above observations based on current results, the following paradigm can bederived concerning the role of radiation, shape of dose response, latency and the radiation
carcinogenesis model. Radiation induces mutations with increasing dose either in a linear orin an LQ fashion. The role of radiation is to contribute only a few mutations such as one or
two to the carcinogenic process. Adult-onset solid tumours require a relatively large numberof mutations, and one mutation by radiation has to be supplemented with additional mutations
from other processes before acquisition of full malignancy. This requires a long latency after
the radiation exposure. The dose response for such cancers follows a linear dose response,
fitting well with the LNT model, and the risk is predicted by the RR model. In contrast,
leukaemia and childhood cancer require much fewer mutations such as two, which can besupplied by radiation alone. For such cancers, the dose-response is LQ with relatively shortlatency, and the associated risk can best be assessed by the AR model. Such characterisation
for the dose response, latency and radiation carcinogenesis model as summarised above isoversimplified, but it offers a foundation for future studies and better understanding of themechanism.

50

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(78) An exception can be found for some cancer types such as radiation-induced
childhood thyroid cancer. This appeared with a short minimum latency of four years after the
Chernobyl accident in the presence of screening programmes. Yet, the dose responses ofchildhood thyroid cancer were reported to be linear (Ron et al., 2012). With the above rule inmind, it is tempting to speculate that childhood thyroid cancer requires two mutations, andthe linear dose response with the short latency occurred for those carrying the pre-existing
RET/PTC rearrangement, with radiation responsible for inducing the second hit necessary forconversion of the cells to full malignancy. Indeed, the rearrangement does exist frequently inbenign nodules in the thyroid, suggesting the possibility of such a mechanism (Marotta et al.,2011).
(79) The choice of risk models for radiation protection has been made empirically whentransferring the risk between two populations differing in the background risk (ICRP, 2007).
A comprehensive review is available on this important subject (Wakeford, 2012). Currently,
the ICRP uses a number of risk transfer models: 1) a mixture of 50% RR model and 50% AR
model for all types of cancer, except for thyroid and skin with 100% RR model, breast andleukaemia with 100% AR model, and lung cancer with a mixture of 30% RR model and 70%
AR model. Among these cancer types, breast cancer with the AR model is clearly an outlierof the above simplified description. Its dose response for women exposed under age 40 islinear with a slight upward curvature instead of expected LQ, the latency is relatively longinstead of just a few years, and the differences in background rates between Japan and Europe
or US are so large that an AR model was considered more prudent and a better fit in agespecific comparisons (Preston et al., 2007). Thus, the working hypothesis as projected here is
too simple to predict the trend of cancer occurrence after radiation exposures. Yet, the present
consideration is useful to gain a possible mechanistic insight of radiation carcinogenesis andits implications for the choice of a risk model for the risk transfer.
(80) The risk and the sensitivity of radiation carcinogenesis are often evaluated by thefunctions EAR and ERR. The EAR quantifies the increment of the cancer incidence rate due
to radiation, while the ERR describes the relative increase over the background (control)
incidence rate due to radiation exposure. Both ERR and EAR not only differ among different
age groups at the time of exposure but also change with time since exposure. ERR is usuallyhigher following exposure at young ages, but the estimates decline with increasing years
since exposure or with increasing attained age. In contrast, EAR estimates at early years since
exposure (i.e. when ERR is highest) are usually smaller and increase along with the increasein years since exposure, because the background rate sharply increases at older ages. Whenattained age is the same, a younger age at exposure in A-bomb survivors may give rise to ahigher EAR for some solid cancer types but not for others.
(81) If it is assumed that cancer occurs as a result of accumulation of certain number ofsomatic mutations, the occurrence of excess cancers as a result of inducing one cancer-related
mutation may be expressed as a function of three factors: (1) the sensitivity of stem cells (andof proximal progenitor or even differentiated cells for certain cancer types) to radiationinduced mutation; (2) the retention of stem cells which had undergone any mutation in any
gene related to cancer development; and (3) the population size of stem cells, at the time of
exposure, which may in the future accumulate a critical number of mutations (say 4
mutations) so that one mutation added by irradiation may result in the elevated rate of cancer.
The number of stem cells with sufficient predisposing mutations is expected to be
proportional to the number with full (say 5) mutations, which results in the backgroundcancer incidence rate. The EAR is obtained by subtracting the background absolute incidence
rate (BAR) from the total absolute incidence rate of cancer (TAR) in an exposed population(EAR = TAR – BAR). In contrast, the excess relative increase in radiosensitivity is quantified
51

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by the ERR which is obtained by dividing the EAR by BAR as in the equation: ERR =EAR/BAR. The EAR depends on the BAR especially for the cancer types where the RR
model provides a better fit when making risk transfer between different populations. A good
example of where the EAR is strongly affected by the BAR can be found in radon-induced
lung cancer in the smoking and non-smoking populations (Darby et al., 2005, 2006). The
EAR was much larger for the smoking population but the ERR was insensitive to thesmoking characteristic. A larger EAR for smokers may reflect a larger BAR of that
population which has an increased number of predisposed target stem cells. The ERR was thesame for both populations. It is tempting to speculate that the smoking-induced conditions did
not markedly affect the sensitivity of target stem cells to radiation induction of carcinogenic
mutations from radon. Altogether, the EAR and ERR functions represent different
mechanistic properties of stem cell behaviour during radiation carcinogenesis (see section
3.6.2).
(82) Representative values of the EAR and ERR functions for some of the tissues
considered in this report are taken from ICRP (2007) and quoted in Table 3.1. These values
are standardised to the risk at attained age 70 following exposure to 1 Gy at age 30. The
uncertainties in the values quoted are highest for breast, lowest for thyroid, with stomach,colon and lung in the middle. The decline in EAR per decade after exposure at age 30 isgreatest for breast, lower and similar for thyroid, stomach and colon, with no change for lung.
The values for ERR are of the same order for all tissues quoted, with declines per decade
after exposure at age 30 being highest for thyroid, lower for stomach and colon, zero forbreast and even a positive increase for lung. The values for bone marrow, skin and bone
surface are not quoted in Table 3.1. because the values are not directly comparable in thesame way. For bone marrow, the preferred dose-response model is LQ (Hsu et al., 2013), andhence the coefficients cannot be compared directly with those in Table 3.1., which are based
solely on a linear model. The EAR dose coefficients for leukaemia at 1 Gy (at age 70 after
exposure at age 30) were 0.70 (linear term) and 0.71 (quadratic term) for women, and 1.06
and 1.09 per 104 PY respectively for men. The corresponding ERR values for men andwomen were 0.79 (linear term) and 0.95 (quadratic term). Of note, compared to decreases forthe other tissues in Table 3.1., the 51% increase in EAR per decade increase in age at
exposure. Skin is a special case, as explained in detail in Annex F, section F.2.1. Risk
coefficients were calculated in a different way from those in Table 3.1. and the values arevery uncertain. For bone cancer incidence in the Life Span Study (LSS) of A-bomb survivors
and assuming a linear dose response, an EAR value of 0.39 (0.08 to 1.04, 90% CI) per 104
PY per Gy for individuals at age 70 exposed at age 30, and an ERR value of 0.48 (0.07 to 1.4,
90% CI), were calculated (Preston et al., 2007). A quadratic model has been suggested(UNSCEAR, 2006), and in addition a more recent analysis indicated that a linear ERR modelwith a threshold at about 0.85 Gy appeared to be the most plausible model from statisticaland biological points of view (Samartzis et al., 2013). A further EAR and LNT model forbone cancer induction has been developed based on 224Ra data (EPA, 2011), although the
LNT model is inconsistent with the 226Ra data where a sigmoid response provides the best fit(Rowland et al., 1978). In view of the above differences and the complexities in making
direct comparisons, in particular for skin and bone versus the other tissues in Table 3.1., amore detailed discussion of EAR and ERR risk values with reference to stem cell-based
mechanisms would be very speculative (see section 3.6.2.).
(83) Values of a tissue weighting factor wT are also quoted in Table 3.1. for completeness,because these formed part of the rationale for the initial choice of tissues for this report (seeIntroduction). The wT is one of the basic elements of the ICRP risk model. It represents therelative contribution of a tissue or organ to the total health risk mainly due to cancer and used
52

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to weight the equivalent dose of such organs (ICRP, 1991). The weighted dose thus derived isthe effective dose from which the nominal risk for a dose can be calculated. The wT values
were revised in 2007 (ICRP, 2007), and again confirmed that the ERR of cancer at age 70 for
people acutely exposed to a unit dose at age 30 years, varies between tissues. The ICRP
recommended a wT value of 0.12 for bone marrow, colon, lung, stomach, breast, 0.04 forbladder, oesophagus, liver and thyroid, and 0.01 for bone surface, brain, salivary glands andskin. The weighting factors refer to total human health risk, i.e. mortality, which is acombination of incidence and the probability that a particular cancer type will be lethal,adjusted for quality of life and years of life lost (see Box A.1, ICRP, 2007). The detrimentEAR
ERR
wT
TissueExcess cases
Age atERR per
Age atTissueper 104 persons exposure: %Gy at age
exposure: %weightingper year perchange in
70 afterchange infactor wT
Gy at age 70
EAR per
exposure at ERR per
after exposure decadeage 30decadeat age 30
increaseincrease
BreastF 10.9-39%
F 0.870
0.12
Thyroid
M 0.69-24%
M 0.53-56%
0.04
F 2.33
F 1.05

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adjusted nominal risk coefficient for cancer is based upon lethality/life-impairment-weighted
data on cancer incidence with adjustment for relative life lost.

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Table 3.1. Coefficients in the cancer-incidence based EAR and ERR models, and tissue
weighting factors, for some of the different tissues considered in this report. Taken from
Tables A.4.7., A.4.6. and A.4.3. in ICRP Publication 103 (ICRP, 2007).
M = Male; F = Female
3.2. Stem cells and stem cell niche in radiation carcinogenesis
3.2.1. Stem cell radiation biology and radiation carcinogenesis

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Stomach
Colon
Lung
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M 6.63F 9.18
M 5.76F 2.40M 6.47F 8.97-24%

M 0.23
F 0.38
M 0.68
F 0.33
M 0.29F 1.36

-24%
+1%

-17%

0.12

-17%

0.12

+17%

0.12

(84) Two factors are essential in carcinogenesis, especially for cancers occurring in
adults. One is the acquisition of oncogenic mutations by the target cells, and the other is theretention of such predisposed cells in the body to allow further accumulation of mutations togain full malignancy. Cellular radiosensitivity and mutagenesis determine the former process,and the dynamics of stem cells in the tissue microenvironment determine the latter.
(85) Radiobiological analyses of pure tissue stem cell populations are still limited.
Nevertheless, past studies and emerging evidence suggest that the radiosensitivity of tissue
stem cells varies considerably between tissues and within a tissue. How these differences
relate to radiosensitivity for carcinogenesis is not clear at present. In the case of the small
intestine, P4 stem cells are highly sensitive to apoptosis by radiation dose as low as 100 mGy
while the telomerase-positive P4 stem cells, putatively the most primitive stem cells of theintestine, survive even 10 Gy of radiation (see paragraph 37 in Chapter 2). This highradioresistance is difficult to understand because after such a dose the cells are unlikely toremain error-free even with extremely efficient DNA repair. This raises a possibility that thetelomerase-positive P4 cells serve as the reserve for emergency purposes, rather than serving
as the house-keeping supplier of lower ranking stem cells. HSCs are one of the mostradiosensitive of all tissues, but the long-term HSCs are more radioresistant than short-term
HSCs (Annex A, paragraph A75). In the skin, stem cells are more radioresistant thanprogenitor cells (Annex F, section F4.1). In general, primitive tissue stem cells are moreradioresistant than their committed counterparts, but the relevance of this to their sensitivity
to radiation carcinogenesis is not known.
(86) Quiescence plays an important role for the radioresistance of stem cells, as
discussed in the previous chapter. In addition, staying quiescent is the best way for tissue
stem cells to avoid replication-mediated mutations and exhaustion of tissue stem cells. At thesame time, quiescence results in accumulation of spontaneous DNA damage as shown inmouse HSCs (Rossi et al., 2007). In addition, quiescent cells have to be dependent on theerror-prone NHEJ repair for coping with DNA damage. Thus, the benefit of quiescence isdependent on the trade-off of avoiding replication-mediated mutation versus taking a chanceof damage accumulation and resulting mutations. Another way to avoid replication-mediatedmutation is the unique mechanism of immortal DNA strand retention and stem cell-specificchromosome segregation, which are likely to operate in stem cells of the intestine andmammary gland. It seems that stem cells have evolved multiple strategies to avoid
replication-mediated mutations. Radiobiological characteristics of quiescent tissue stem cells
in vivo need to be further analysed in order to understand the cellular processes of radiation
carcinogenesis.
3.2.2. Tissue radiation biology and radiation carcinogenesis(87) The steady-state maintenance of a tissue involves three tissue compartments: thestem cell compartment, the progenitor compartment and the functional cell compartment.
Once cells move out of the first compartment, they will be discarded eventually from thebody, except for some progenitors of long residential time in a tissue. Thus, stem cells are the54DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
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major target with long enough time of residence in the body to accumulate mutations andthus acquire a malignant phenotype. However, as discussed in section 3.1.2, even stem cells
are not immune to being replaced by other stem cells since they are under constantcompetition for residence in the stem cell niche. This competition is due to occasional
symmetric division of tissue stem cells in which extra stem cells compete for the residence inthe niche.
(88) Analyses of Drosophilla germ cells revealed E-cadherin as the major gene involvedin the competitiveness of the germline stem cells in the niche (Zhao and Xi, 2010). In thisDrosophilla system, rapid turnover of stem cells functions as an efficient mechanism for theremoval of aberrant stem cells in the gonad. This is likely to be so for mammalian stem cellsystems where stem cells are in contact with various niche cells. HSCs interact withosteoblasts through N-cadherin and integrin, and receive the Tie2/Ang-1 signalling toregulate the quiescence (Suda, 2007). As for Lgr5+ stem cells in the small intestine, adhesion
to Paneth cells was found to be essential for keeping the stemness of the cells throughessential signals received for their maintenance (Sato et al., 2011). Change in the expressionof these genes, brought about by any stress including radiation and/or by mutations inrelevant genes, is likely to affect the competitiveness of stem cells and their subsequent
occupancy in the niche. The effect of low dose radiation on the niche interaction of stem cells
has not been studied. Expression of E-cadherin was studied at >2 Gy with contradicting
results; downregulation in mammary epithelial cells and upregulation in the rat liver(Andarawewa et al., 2007; Moriconi et al., 2009). The consequence of such changes is
expected to result in the less-fit stem cells being competed out of the niche. Indeed, it wasreported that irradiation of bone marrow with >0.5 Gy decreased the competitiveness of
HSCs in the recipient hosts (Marusyk et al., 2010). This tissue microenvironment-based
selection process is likely to function as a tissue-based quality control, independently fromthe molecular and cellular-based quality controls such as DNA repair and apoptosis.

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3.3. Dose rate effect for radiation carcinogenesis

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3.3.1. Cell-based considerations(89) For radiological protection, the risk of radiation has to be estimated especially forlow dose and low dose rate exposures, where the risk to be taken into consideration is that forstochastic effects (ICRP, 2007). The ICRP estimates such risk by the use of the LNT modeland DDREF to adjust for the conditions of low dose and low dose rate exposures. The ICRP
made a choice of 2.0 for the value of the DDREF and is continuing to use it (ICRP, 1991;
ICRP, 2007). The dose response of cancer for acute exposures generally follows an upward
concave curve which can be adequately described by a number of models. Two of the cellbased biophysical models have been well received: the sublesion model of Kellerer and Rossi(1972) and the repair saturation model of Goodhead (1985). In particular, the sublesion
model is widely used with an LQ equation as below (UNSCEAR, 2006):
E (D) = αD + βD2(1)(90) The linear term of the equation (1) represents single-track events in cells which aresupposed to be dose-rate independent. The quadratic term represents two-track events which
are subject to cellular repair, and therefore this term becomes negligible at low dose and at
low dose rate. As for the definition of low dose, any doses below 200 mSv were proposed by
UNSCEAR since in this dose range, the linear term dominates the magnitude of the dose55

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response (UNSCEAR, 1993). In addition, under the assumption of equation (1), the risk ofradiation carcinogenesis becomes identical for low dose and low dose rate. This is the reason
why the low dose and low dose rate can be handled by one factor of DDREF. Then, DDREF
can be described by the following equation:
DDREF = (αD + βD2) / αD = 1 + (β/α) D(2)(91) The US Biological Effects of Ionizing Radiation (BEIR) VII Committee applied
equation (2) to the epidemiological data of the A-bomb survivors and animal data using a
Bayesian approach, and obtained a DDREF value of 1.5 (BEIR VII, 2006). Furthermore,studies of radiation workers exposed at low dose rates reported similar risk coefficients forsolid cancer as those observed for acute exposures in the LSS, suggesting a DDREF value of
1.0 (Jacob et al., 2009). UNSCEAR abandoned the use of DDREF and applied the LQ modelto the LSS data to directly obtain the low-dose/low-dose-rate risk coefficient (UNSCEAR,
2006). The risk coefficient thus obtained was consistent with the ICRP risk values based on
the LSS data adjusted by the DDREF value of 2.0.
(92) The calculation-based derivation of the DDREF by BEIR VII and UNSCEAR(UNSCEAR, 2006) relies on the validity of the LQ equation, and especially on the dose-rate
independence of the linear term. At the cellular level, there is ample evidence that the linear
term of the mutation induction rate by radiation is independent of dose rate. However, at thetissue level, there are cases in which the slope of the linear dose response of mutation andcancer decreases by lowering the dose rate. Such a decrease can be expected when the tissuelevel elimination of aberrant cells by stem cell competition is in operation.
3.3.2. Tissue-based considerations(93) As discussed in the Chapter 2, HSCs of mice irradiated with ≥1 Gy are effectively
competed out by those of unirradiated mice as demonstrated by co-transplantation into
lethally-irradiated mice (Bondar and Medzhitov, 2010). This suggests that any deviation fromtotal stemness by irradiation leads to elimination of such cells from the tissue. Stem cellcompetition is regarded as a quality control at the tissue level to eliminate phenotypicallyunfit cells. Thus, there are three levels of quality control systems in a body: the molecularlevel quality control of DNA repair; the cellular level quality control of apoptosis; and thetissue level quality control of stem cell competition.
(94) Stem cell elimination is expected to affect the linear term of the LQ equation (1).
One example of such case is seen in the radiation induction of germline mutations in mice. In
the case of the male mouse, the induction is a linear function of radiation dose, yet theinduction rate was lowered when the dose rate was reduced (Russell and Kelly, 1982). In thecase of the female mouse, the dose-rate effect is extreme in that the linear dose response
became completely flat, when the dose rate was decreased (Searle, 1974). Molecular andcellular quality controls cannot explain such data. As discussed in the previous section, thelow dose-rate sparing of mutation by DNA repair should affect only the quadratic term of theLQ equation, and the linear term should not be affected by the dose rate. In addition, cellularquality control of apoptosis functions is less efficient when the dose rate becomes low. Thus,
loss of the irradiated cells by whatever mechanism is likely to contribute not to the DEF, but
to the DREF.
(95) Key issues in this topic are whether low dose exposure influences the stemness andif it does, how low can be the dose to affect the stemness. If the dose is as low as anelemental dose of radiation, that is, the lowest dose given by a single track of radiation to a56

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nucleus of a cell, then an interesting possibility emerges. An elemental dose of 60Co γ rays isaround 1 mGy for the typical mammalian cellular nucleus of 8 µm in diameter (Feinendegen,1985). Chronic exposures at a dose rate of a few mGy per year mean that every cell in the
body is hit by a track of radiation every few months. This then makes a hit stem cell, at any
time, compete against surrounding non-hit stem cells within a niche. Thus, if the elemental
dose affects the stemness, the hit cell will be preferentially lost by competition from the tissue
stem cell niche. This elimination theory lowers the linear term. Hence, stem cell competitionat the tissue level leaves an ample possibility for a DREF value larger than unity, as in thecase of the current DDREF value used by the ICRP.

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3.4. Experimental animal studies to supplement human data

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(96) There have been many studies of radiation induced tumours in animal systems whichhave contributed knowledge of dose-incidence relationships and dose-rate/fractionation
effects. These have focussed mainly on AML and solid tumours of the Harderian gland,
pituitary, ovary, lung, breast, skin, and bone (ICRP, 2006; NCRP, 2005). Overall, it wasconsidered by the Commission that the animal tumour data tended to support the hypothesisof a linear relationship of incidence versus dose at low doses and at low dose rates, with no
threshold dose (ICRP, 2005). However, there are various caveats to this conclusion, notablythat some of the tumour types studied in experimental animals are not the most relevant types
in humans, and there are many strain and species differences in sensitivity. In addition, over abroad range of doses, all neoplasms are not increased in frequency within a given species or
strains and certain neoplasms are decreased in frequency by irradiation. It has been argued
that “From the diversity of observed dose-incidence relationships, it is clear that no one
mathematical model for relating incidence to dose is universally applicable” (Upton, 1985).
Nonetheless, the study of radiation induced tumours in one or more defined animal strains
can help elucidate the molecular, cellular and tissue mechanisms which are fundamental to
any animal species including humans.
(97) Concerning radiation-induced stochastic events in the haematopoietic system(Annex A), AML is the type of leukaemia in mice which has been studied in most detail.
AML comprises less than 5% of all childhood leukaemias, but is one of three radiationinducible leukaemia subtypes: AML; chronic myeloid leukaemia (CML); and acutelymphoblastic leukaemia (ALL). Regarding the target cells for AML, recent evidence in micesuggests that the initial radiation-induced AML stem cell may originate not only from
irradiated HSCs but also from multipotent progenitor cells and common myeloid progenitors(Hirouchi et al., 2011; and Section A.5). Also, hemizygous deletion of Dusp2 in chromosome
2 may contribute to the self-renewal potential of radiation-induced AML stem cells. A
detailed study of the “immortal strand hypothesis” in highly purified HSCs revealed that
BrdU had poor specificity and poor sensitivity as an HSC marker (Kiel et al., 2007). All HSC
segregated their chromosomes randomly, and division of individual HSC in culture revealed
no asymmetric segregation of the label. Hence, HSCs did not retain older DNA strands
during division. The multistage theory of carcinogenesis and the importance of the
microenvironment in promotional events continue to suggest that the target cells are likely tobe the more slowly renewing cells in the lineage, and those cells are generally the moreprimitive stem cells in the population. Indeed, the correlation between chronic radionuclide
doses/location and AML incidence was closest for target cells in the central marrowsinusoidal region (Lord et al., 2001), which is a principal site of such primitive cells. These
are identified in marker studies as CD34-CD48-CD150hiLSK.

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(98) As noted in Section A.1.1, the incidence of AML at very low levels of exposure(from background levels to approximately 1 Gy) is fairly linear with increasing dose. At
doses from 1 to 2-3 Gy, the dose-incidence curve tends to exhibit upward curvature, and at>3 Gy, the curve tends to bend over downwards. Also, DDREF values estimated from suchdata vary quite a lot, but generally fall in the range of 2 to 5 over that dose range (UNSCEAR,
1993). Values at smaller doses are expected to be lower, reflecting mainly the single-hit (αcomponent) of the dose-response curve. In often-quoted mouse experiments deliveringcontinuous irradiation for 28 days at 0.04-0.11 mGy/min to a total dose of 1.5 Gy, the AML
incidence of 5% (Mole et al., 1983) was also produced by an acute dose of about 0.5 Gy(Mole and Major, 1983), which gives a DREF value of ≤3 at ≤1.5 Gy. In addition, after
higher doses of 3.0 and 4.5 Gy delivered at the same low dose rate, the AML incidence wasinexplicably also 5%. This led to the postulate of some “biological factors” related tostem/clonogenic cells, influencing the response to dose protraction (Mole and Major, 1983),
as discussed further in section A.2.2.
(99) A number of studies have been published on the effects of dose and dose rate on
mammary tumour induction in rodents (Annex B; UNSCEAR, 1993). Studies with SpragueDawley rats gave an approximately-linear incidence with increasing dose. The DDREF
values ranged from less than 2 to about 4, for dose rates varying by a factor of ≥150 and for
doses (at high dose rate) between about 2 to 3 Gy (Shellabarger et al., 1966; Gragtmans et al.,1984). With BALB/c mice, the dose-incidence curve at high dose rate was LQ up to about
0.25 Gy, and the linear term was similar to that obtained after low dose rate (0.07 mGy/min)
exposures (Ullrich, 1983). Dose-fractionation studies showed a significant contribution fromthe quadratic component at doses as low as 0.1 Gy/fraction, and acute daily fractions of 0.01
Gy gave a tumour incidence similar to that observed after the low-dose-rate exposure to a
total dose of 0.25 Gy in both cases (Ullrich et al., 1987). The DDREF decreased withdecreasing dose from 11.7 at 0.25 Gy, and would be predicted to be near unity at a dose of
between 0.1 and 0.01 Gy. The mouse studies showed that dose fractionation/protraction to 25
days (0.25/0.01) produced the incidence of mammary cancer predicted from the LQ analysisof the acute exposure response.
(100) Regarding age-at-exposure effects, the relatively restricted window of carcinogensusceptibility that is evident during or around puberty in both rodents and humans has been
postulated to either contain the greatest number of target cells or be a critical period of stem
cell regulation. There is a clear hierarchical lineage in mammary epithelium, and the manyfactors that control it. For example, the CD24+ CD29high population in the mouse mammary
epithelium is highly enriched for cells with multilineage and self-renewal potentials, the two
properties that define a MaSC. Also, there is evidence that epithelial LRCs in mouse
mammary gland divide asymmetrically and retain their template DNA strands (Smith, 2005).
However, the target cell origin of radiation-induced breast cancers in terms of stem andprogenitor cells is not yet elucidated.
(101) Concerning the thyroid (Annex C), radiation induces both papillary and follicular
carcinomas, but in humans the former type predominates whereas in the common rat model
the latter type predominates. Dose-incidence relationships for carcinomas (mostly follicular)
in 3000 female Long-Evans rats showed a rising incidence with increasing x-ray dose from
0.8 Gy, flattening off at the higher doses to 10.6 Gy (Lee et al., 1982). However, adenomas
were in the majority, and in contrast these showed a continuously rising dose-incidence curve.
Hence the curves for the two tumour types appeared to be significantly different in shape. In
addition, concurrent studies with 131I and detailed dosimetry (Lee et al., 1979), showed asimilar response to the high-dose-rate x-ray results for the carcinomas, but there was atendency towards a lower incidence of adenomas at the higher doses of 131I compared to x58

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rays. If the adenoma yields are interpreted on an LQ basis, it can be estimated that the solelylinear component (not modified by dose rate) may be at doses up to about the first dose pointof 0.8 Gy, and a DDREF of around 2 may apply at about 2 Gy. However, for the aggregatedyields of both tumour types, the solely-linear component could be higher and the DDREF
lower, albeit with large uncertainties. The similarity of tumour yields in the rats at low dosesof acute x-rays and low dose rate 131I is compatible with the human data, i.e. the ERR for
external radiation exposure was compatible with the ERR estimates for internal radiation
exposure following the Chernobyl accident (section C.2). Although there is evidence for thepresence of a stem cell-type lineage in the thyroid epithelium (section C.3), there is noknowledge of whether the different tumour types originate from the same or different target
cells in the lineage.
(102) Cancers of the stomach and colon (Annex D) in rodents are induced only by highradiation doses, e.g. ≥8 Gy (Boice and Fry, 1995), and are only very rarely found in the small
intestine. A possible reason invoked for the latter is the radiation-induced apoptosis ofmutated stem cells in the small intestine, which is prevented in the large bowel by theexpression of the survival (anti-apoptotic) gene bcl-2 (Merritt et al., 1995). Nonetheless, thestomach and colon are fairly resistant to cancer induction. The Min mouse provides asensitive model for the study of tumourigenesis in irradiated mice. Min mice are geneticallyheterozygous for a germline truncating mutation of the Apc gene (i.e. ApcMin/+) and develop
multiple intestinal tumours and sporadic colon tumours in their intestinal tracts within several
weeks of birth. The following yields of tumours were observed in CHB6 ApcMin/+ mice
exposed to 2 Gy x-rays in utero on day 7 (30 tumours/mouse, not significantly raised above
numbers in unirradiated controls) or day 14 post-conception (44 tumours/mouse), as 2-day
neonates (85/mouse) or as 10-day neonates (130/mouse), and 35-day young adults(70/mouse) (Ellender et al., 2006). Hence, neonates were more sensitive to tumour induction
than young adults. The x-ray dose-incidence curve for adenomas was LQ over the range 0-5
Gy, and strikingly there were more tumours in the small intestine than in the caecum andcolorectum (Ellender et al., 2011). In the caecum, the tumour incidence was elevated after ≥2
Gy, and in the colorectum after ≥1 Gy. In general, adenomas in the small intestine weresessile while the smaller numbers of adenomas in the large intestine were pedunculated.
There was also an incidence of microadenomas in the small intestine, which was greater after
the higher doses in the range used, but none was found in the large intestine.
(103) The target cell for colonic tumours is still considered to be the crypt stem cells, andthe potential inclusion of progenitor cells as target cells is not yet resolved. An interestingdevelopment is the finding of rare (1 per 150 crypts), slowly-cycling, long-lived andradioresistant mTert+ stem cells in both small and large intestine, giving rise to alldifferentiated intestinal cell types. These are probably the best candidate target cells in thecolon in terms of the multistage model for carcinogenesis. Germline mutation of the APC
gene predisposes both humans and mice to intestinal carcinogenesis. In humans, inheritanceof mutant APC is associated with the cancer predisposing disorder, familial adenomatous
polyposis (FAP), and mutation of APC is an early somatic event in sporadic colon cancer.
Individuals carrying germline mutations in the APC gene develop hundreds to thousands ofcolorectal adenomatous polyps, some of which will progress to carcinomas if left untreated.
(104) From studies of A-bomb survivors and uranium miners, radiation-induced lungcancers (Annex E) appeared to be more likely small-cell lung carcinomas (SCLCs), and lesslikely to be ADCs (Land et al., 1993). In mice, SCLC does not occur, and ADCs are the mostcommon type of lung cancer. The induction of ADCs in female BALB/c mice following
acute irradiation was shown to be consistent with an LQ model in which the linear term was
independent of dose rate, by comparing responses using 0.4 Gy/min and 0.06 mGy/min
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(Ullrich, 1983). Also, the DDREF was about 4.2 at 3 Gy, and about 3.2 at 2 Gy, and fromdose fractionation studies, the DDREF was predicted to be about 1.1 at 0.1 Gy (Ullrich et al.,1987). Like for mammary tumours induced in this same strain of mouse, the induced
frequencies of lung tumours from acute, and protracted fractionated or low-dose-rate
exposures, were consistent with each other in an LQ analysis.
(105) In the respiratory tract, the target cells for radiation-associated carcinogenesis are
considered to be in the basal cells in the trachea and larger bronchi of the central lung, and in
the Clara variant and type II alveolar cells of the peripheral lung (Annex E). An epithelial
stem cell niche has been identified in the zone where airways terminate and form alveoli. The
putative mouse BASCs in the bronchiolar alveolar junction, coexpress secretoglobin a1a(SCGBa1a), the type II cell marker surfactant protein C, Sca-1, and are negative for CD45
and CD31. Molecular analysis showed that, despite their distinct histopathologicalphenotypes, in human ADC and SCC, genomic profiles showed a nearly complete overlap,
with only one clear SCC-specific amplicon (Tonon et al., 2005). Hence the common ordifferent cellular origin of lung cancer types may become better understood. In addition, there
may be influences from the irradiated microenvironment. For example, migration of MSCs
into irradiated and stressed regions has been invoked as a potential alternative or contributory
mechanism in carcinogenesis.
(106) Radiation induced skin cancers (Annex F) in humans are predominantly BCC. The
traditional view was that a threshold dose exists for radiation-induced skin cancer, in therange of 8 to 10 Gy, but the A-bomb survivor data indicated that BCC can be induced byacute exposure at moderate doses, in the range of 1-4 Gy. In mice, radiation readily produces
SCC but no BCC, whereas in rats about 20% of induced skin tumours are BCC. The doseresponse curve for total tumours in rats was compatible with an LQ model (and linear forhigh LET radiation), albeit with a tendency for adnexal (hair follicle and sebaceous) tumours
to be more common and later after low doses compared to epidermoid tumours being morecommon and earlier after high doses. With repeated weekly doses of 0.75 or 1.5 Gy over a
lifetime, more tumours were produced than expected from single-dose responses, suggesting
either the number of events increased per unit dose (i.e. induced sensitisation) or that clonal
growth expanded the number of early transformed cells (Burns and Albert, 1986a and 1986b).
The radiation had to penetrate at least about 180 µm to induce tumours, and 300 µm wasabout optimum irrespective of follicle growth phase and size, so demonstrating that the main
target cells were in the stem cell zone of the hair follicle. Also, there was a marked effect ofage on radiation-induced cancer incidence. In the rat, the dose to induce 2 tumours per rat(the mid-range induced) by 70 weeks postirradiation increased from 10 Gy (1 day old) to 15
Gy (28 days), 17.5 Gy (58 days) and 30 Gy (99 days). If this increasing resistance isexpressed as an iso-effect per unit area of skin irradiated, the factor of 3 in iso-effective dose
per rat between 1 and 99 days of age would be greater by likely 2 orders of magnitude (i.e. 3
x ~100), when allowing for the smaller skin area of the irradiated newborn compared to themuch larger adult rat.
(107) A model for human skin cancer proposed that stem cells were likely target cells for
BCC, early progenitor cells for SCC, and late progenitor cells for papillomas (Sell, 2004).
Molecular characterisation of these different cell populations is continuing, and the mostpotent quiescent stem cells appear to be marked as α6briCD71dim. There are quantitative dataof mouse follicle-bulge cell divisions marked with BrdU, which support the long-standing
infrequent SC-division model (Waghmare et al., 2008). However, it was shown that hairfollicle stem cells do not retain the older DNA strands or sort their chromosomes. To date,there are no distinct markers for the target cells of the different types of skin cancer.

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(108) Radiation-induced bone sarcoma has been associated with high doses of ionisingradiation from therapeutic or occupation-related exposures (Annex G). However, thedevelopment of bone sarcoma following lower doses remains speculative. Analysis of 80,000
individuals in the LSS cohort to assess the development of bone sarcoma (most commonly
osteosarcoma) in A-bomb survivors, showed a preferred fit with a dose threshold at ~0.85 Gy(95% CI, 0.12 to 1.85 Gy) and a linear dose-response association above this threshold(Samartzis et al., 2011). Chadwick et al. (1995) fitted the radium dial painter data using a
two-mutation carcinogenic model with clonal expansion (Chadwick et al., 1995). The
analysis showed that an LQ dose-effect relationship can be applied and, because of the very
low natural incidence of bone sarcoma, is consistent with very low AR at low doses and dose
rates. Much of the experimental work on radiation-induced bone cancers has been performed
using dogs. For the (low-LET) β emitter, 90Sr, the dose response was non-linear with notumours occurring at doses below 18 Gy cumulative average bone dose (Annex G). This
much higher threshold dose may reflect differences in dosimetry, the protraction of theradionuclide dose, and the much shorter lifespan of dogs versus humans. Mesenchymal(osteogenic) stem cells (MSCs) for the osteoblast lineage reside in the bone marrow. CD34negative stem cells as well as mesenchymal precursors are possible target cells for radiationinduced bone cancer. In mice, these target cells for bone-associated α-emitters lie within
about 40 µm from the endosteal surface.

2500	
  

3.5. Location of target cells in a tissue

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(109) The location of target cells in different tissues is an important consideration in thecalculation of doses received from short-range particulate emissions from radionuclides
retained in body tissues, including α particles and low energy electrons. Thus, theCommission has made some judgements and assumptions about the location of target cells inthe skin, the respiratory and alimentary tracts, and the skeleton (ICRP, 1991, 1995, 1996,2007). Based on the data in the present report and recent publications, the location andcharacteristics of target cells are assembled in Table 3.2.
(110) For tissues where the stem cell location is understood and well-defined, it may bereasonable to estimate doses specifically to this location since stem cells are the primarytarget for accumulation of mutations in the initiation and development of carcinogenesis.
However, the extent to which the immediate progeny of stem cells may also be targets for thedevelopment of particular cancer types varies between tissues and is not well established inmany cases. For some tissues, this possibility may not have implications for the definition oftargets for dosimetric purposes because stem cells and their immediate progeny occupy thesame microenvironment. For example, in the epidermis, the stem cells and immediatedaughter cells are found in the basal layer. The nominal depth of target cells in humanepidermis is taken to be 70 µm for dosimetric protection calculations, although there issignificant variation in depth of the skin undulations. For the example of the colon, thedistance from the mucosal surface is taken to be the crypt base at 280–300 µm depth, andwidening this band to include progeny cells makes little difference to dosimetric calculations
when considering irradiation predominantly from radionuclides in the intestinal lumen. For
the respiratory tract, however, assumptions regarding the type and location of target cells
within the airways can be a major determinant of estimated doses for some radionuclides,
including the α particle emitting progeny of radon-222. For bone cancer, until recently thetarget has been taken to be a 10 µm layer adjacent to bone surfaces, but now it is recognisedthat a 40-50 µm layer would be more appropriate. For leukaemia, while it is known that stem
cells are located in endosteal and vascular niches, refinement from the calculation of average

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red bone marrow dose has not proved feasible, although it is recognised that risks ofleukaemia from bone-seeking α particle emitters (plutonium-239, radium-226) aresubstantially overestimated by such calculations. For other tissues such as mammary glandand thyroid, regional distributions of potential stem cells are not considered, and radiation
doses are calculated as tissue averages.
Table 3.2. Locations and characteristics of target cells for radiation-induced cancers in different
tissues.
Annex, Cancer Human target cells
Markers
LocationA, Leukaemia

HSCs (and potentiallysome progenitor cells)

CD34+, CD59+, Thy1+,
CD38low/-, c-Kit-/low, Lin-

Endosteal andvascular niches

B, Breast

Mammary stem cells

Possibly CD24+
CD29high, and possibly
K6

Mammary MaSC
niche (not welldefined)C, Thyroid

Follicular stem cells

Possibly Oct4+Pax8+Tg–

Solid cell nests(SCNs)

D, Stomach

Mucosal stem cells

Possible LRC(defensin5-, Muc2-,
chromograninA-)

Gastric pits, 60–
100 µm depth

D, Colon

Mucosal stem cells(possibly also somedaughter cells)

Lgr5+; mTert+; possibly
also DCAMKL-1

Crypt base, 280–
300 µm depth

E, Lung

Possibly Clara, Claravariant, or BASC cells

SCGBa1a, surfactant
protein C, Sca-1, CD45, CD31-; also possibly(c-KIT+, Nanog, Oct ¾,
KLF4, Sox2) cells

Bronchiolar–
alveolar duct
junction zone, also
possible distallung niche

F, Skin

EpiSCs – BCC (alsoearly progenitors – SCC,
late progenitors –papillomas)

α6briCD71dim; alsoβ6bri/CD71dim

Interfollicularbasal layer,
nominal 70 µm
depthG, Bone

MSCs

CD90, CD73, CD105,
and possibly Stro-1,
CD106, VCAM-1

MSC niches andperivascular

CD34-

Bone marrow

Some HSCs
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3.6. Cell-based and tissue-based considerations

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(111) Regarding the relationship between the cancer incidence parameters (Table 3.1.)
and the three considered mechanistic factors of (1) the number and sensitivity of stem cells toradiation induced mutation, (2) the retention of mutated stem cells in a tissue, and (3) the
population size of stem cells with a sufficient number of predisposing mutations, there is a62

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lack of definitive evidence for the contribution of one or more factors to the values ofradiation risk for the different tissues. One example is that on current estimates, differences inthe stem cell number among tissues are unlikely to be an important factor, albeit with the
caveat of large uncertainties in the numbers. Some estimates of total stem cells per human inbone marrow are ~108 (Annex A), colon ~4 x107 (Annex D), and ~2 x107 for skin (Annex F;
1 functional stem cell per 35,000 epidermal cells, and 8 x1010 total epidermal cells perhuman). These numbers span more than 3 orders of magnitude, and their ranking clearly does
not show a similar ranking of sensitivities regarding cancer incidence expressed by either
EAR or ERR functions. An extreme example is the small intestine with estimated more stem
cells (~2x108) than in the colon (~4x107), and yet the risk of radiation-induced cancer in thesmall intestine is virtually zero compared to positive values in the colon. Biological reasonsfor this have been proposed based on more p53-mediated apoptosis in the stem cell zone in
the small intestine, compared to its prevention by bcl-2 expression in the colon. Also there
may be differences in the true target cell population in the two sites, depending on whether it
includes extra ‘potential’ stem cells and hence is larger, or whether it consists of a minority ofradioresistant mTert+ stem cells which differs between sites and hence forms a smaller
population.
(112) Mechanisms behind any tissue differences in the sensitivity to radiation
carcinogenesis are likely to be multiple, ranging from tissue specific mechanisms of stem-cellnumber and turnover, and cell-type specific mechanisms of DNA replication, DNA repair,cell cycle control and apoptosis. Knowledge in these areas is still lacking, especially for thetissue stem cells. One general trend is that the risk of radiation carcinogenesis is higher forsome tissues with higher rates of renewal such as skin, colon and stomach, while lower for
those with lower rates of renewal such as oesophagus, liver, thyroid, and bone surfaces,
although there are exceptions, e.g. bone marrow. It has been thought that a higher rate of stem
cell proliferation could contribute to a more rapid rate of mutation accumulation, but also ahigher turnover rate of progenitor cells may not allow time for the cells to accumulate enoughmutations to acquire full malignancy. Adult tissues without proliferation are almost refractory
to radiation carcinogenesis and brain can be such an example, although a small number of
NSCs are slowly proliferating even in adult primate brain (Gould et al., 1999). In contrast,some highly sensitive tissues like breast are not particularly active in proliferation (Annex B).
Similarly, bladder is known to be sensitive to radiation carcinogenesis, yet this tissue is rather
quiescent. Thus, simple proliferative activity of tissues does not appear to predict tissue
sensitivity to radiation carcinogenesis.
(113) Some tissue stem cells are characterised by specific mechanisms such asasymmetric segregation of template strands when replicating DNA, use of a specific DNA
repair system such as NHEJ, altruistic apoptotic cell death and cellular differentiation. These
cellular features are tightly regulated by the tissue microenvironment of the stem cell niche,
which is essential in maintaining the stemness of the stem cells. Thus, any perturbation inthese features is likely to lead to accumulation of mutations. In the case of mammary gland,
the niche was shown to be the target of radiation carcinogenesis (Section B.4.2). For sometissues, stem cells are under constant competition for the occupancy of the niche, which leads
to elimination of some of its stem cells. This elimination can be a contributing factor for thetissue-specific sensitivity to radiation. It is concluded that a number of mechanisms are
known or speculated upon in this report which may contribute in some way to cancer
susceptibility among tissues, but to date their applicability and importance are unknown.

63

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3.7. Age dependence of radiation carcinogenesis
3.7.1. Age dependent occurrence of spontaneous cancers(114) Childhood cancer is defined as those occurring after birth until puberty, andsupposed to arise in children carrying predisposing mutations inherited from their parent. The
mutation may also be acquired during fetal development or childhood growth. The incidenceof childhood cancer is roughly 1 x 10-4 per live birth. The type of childhood cancer isrestricted, and each cancer type has a specific age window of occurrence (Ries et al., 1999).
For example, retinoblastoma of the hereditary form develops within a year after birth. In
addition, these carriers develop bone sarcoma around the time of adolescence (Knudson,
1971; Abramson et al., 1984; Friend et al., 1986). This bimodal pattern of occurrence isshared by the non-hereditary form of retinoblastoma and bone sarcoma as well (Ries et al.,1999).
(115) Adult cancers are supposed to occur as the result of mutations acquired somatically,and exhibit a steady increase in the incidence with age (Armitage and Doll, 1954). This agedependent increase makes cancer the leading cause of death in developed countries with along life expectancy. The incidence of cancer differs by gender. It is slightly higher for males
up to adolescence and almost twice as high for males than females after 70 years of age(Bleyer et al., 2006). The incidence of adulthood cancer is twice as high in females as inmales in their 40s due to the female-specific cancers in this age group.
3.7.2. Risks from fetal-stage radiation exposures(116) Russell and Russell (1954) were the first to show a strong developmental-stage
dependence of radiation effects in mice. They exposed pregnant female mice to radiation, andfound that the pre-implantation stage was highly sensitive to embryonic death, but nomalformation was identified. The fetal stage was sensitive to radiation induction of
malformations, but the occurrence of cancer was not reported. Another classic experiment
performed by Bissell and her group noted that chick embryos lacked tumour formation evenwhen inoculated with highly oncogenic Rous sarcoma virus, suggesting strong suppression oftransformed phenotype in embryos (Howlett et al., 1988). Interestingly, such chicks after
hatching developed tumours at the site where the injury occurred. Based on these classic
studies, it is tempting to speculate that cancer is the disease taking place in the establishedadult-type tissue architecture including the tissue stem-cell niche.
(117) One of the largest human studies on the effect of fetal stage exposures was the
Oxford Survey of Childhood Cancers (OSCC). OSCC, a case/control study of mortality fromchildhood cancer in Great Britain found an association with an intra-uterine x-ray
examination, and indicated that fetal stages were highly sensitive to radiation with an ERR
per Gy of 50 for childhood leukaemia and other childhood cancers alike (Wakeford and Little,
2003). The epidemiological study of the incidence of cancer among the in utero exposed Abomb survivors suggested a high ERR for childhood cancers other than leukaemia of 22 perGy (although based upon just two incident cases and was thus not statistically meaningful),
but no raised risk of childhood leukaemia (Wakeford and Little, 2003). The study of Ohtaki
et al. (2004) of chromosome translocations in peripheral blood lymphocytes sampled from Abomb survivors exposed in utero found no dose response above a dose of around 100 mGy,in contrast to the dose response found in some of the mothers and other adults. The authors
suggested the lack of chromosome aberrations being due to a high sensitivity of the fetal
stage haematopoietic cells to killing by radiation at moderate doses, i.e. doses much lower

64

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than those normally associated with cell killing affecting cancer risks. Competition-mediatedelimination of stem cells from newly established bone marrow niches may also explain thelack of chromosome aberrations in lymphocytes of in utero exposed A-bomb survivors. A
moderate ERR of 1.0 per Gy was found for mainly adult-onset solid tumours, with an overallrisk lower than that for the childhood exposures with an ERR value of 1.7 (Preston et al.,2008). It is interesting to note that a combined analysis of the in utero exposed and thechildhood-exposed indicated a dose-response of upward curvature, suggesting a quadraticcomponent in the induction of cancer for these cohorts. The Commission made an extensive
review, but at that time was unable to reach a clear-cut conclusion on the risks of fetal stage
exposures (ICRP, 2003). Based on the data available, and uncertainty on the development ofsolid cancer for the in utero exposed, the ICRP made a judgment that the life-time cancer risk
following in utero exposure is similar to that following irradiation in early childhood (ICRP,
2007). Based on more recent follow-up, this assumption appears to overestimate the lifetime
risk of in utero exposure (Preston et al., 2008).
(118) Ideally, the unresolved issue of the epidemiological studies has to be augmented
with the help of experimental studies. However, one problem of such an experimental
approach is the lack of an appropriate animal model of human childhood cancer. For example,
human childhood cancer is relatively rare with the cumulative incidence of roughly 10-4 frombirth to 15 years of age. Experimental studies usually utilise a group of less than 100 animals
which are too small to detect cancers arising at a frequency of 10-4. Therefore, experimental
studies are conducted to analyse the effects of in utero exposures on the lifetime occurrenceof cancer, which mimics adult-onset cancer in humans. Such studies using laboratory miceand rats demonstrated that in utero exposures are less effective than neonatal exposures in
inducing leukaemia and various solid tumours (Upton et al., 1960; Sasaki, 1991; Inano et al.,
1996; Di Majo et al., 2003). Detailed studies on the age-dependent sensitivity of the ApcMin/+model mice to radiation, indicated that the sensitivity was highest for 10 day-old neonatesand decreased in the order of 2 day old neonates, 35 day young adults, 14 day fetuses and 7
day embryos (Ellender et al., 2006). Mouse studies thus indicate that the fetal stage in generalis less sensitive than neonates, and that the earlier embryonic stage is much less sensitive toradiation induction of leukaemia and solid tumours. The fetal stage has a shorter duration inmouse than in man and this could be one of the reasons for the different results between these
two species. The OSCC reported that the first trimester was most sensitive to induction ofchildhood cancer (Bithell and Stiller, 1988), and this is in contradiction to the mouse data
where the early embryogenesis stages are generally insensitive to radiation carcinogenesis.
One possible reason is that the doses to the first trimester could have been greater than thosereceived during the second and third trimesters (Mole, 1990), and the apparent greater
sensitivity could just be related to the dose received during an examination (Doll andWakeford, 1997). It is important to note that none of the case-control studies of prenatal
exposure and childhood cancer risk performed individual dose reconstructions on the cases
and the controls involved. Dose estimates were based on national surveys and there areuncertainties with regard to machine parameters, repeat examinations, and undocumented
procedures.
(119) When considering cellular characteristics, it is difficult to discern the difference
between stem cells of fetal stages and after birth. Therefore, the difference in the sensitivity
to radiation carcinogenesis has to be sought at the tissue level. One distinction of fetal stage
tissues is the lack of a clear niche-like microarchitecture while in the adult, tissue stem cells
are thought to reside in a distinct microenvironment of a stem-cell niche. The adult-typeniche is established after birth for many tissues. As discussed in Chapter 2 (section 2.5.4) for
example, a major site of HSC proliferation is the liver during fetal development. HSCs then65

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migrate and colonise the bone marrow niche. Numerous HSCs migrate to the newly
established bone marrow niche housing a limited number, and it was shown that the only
HSCs which are able to settle in the niche are those in the G0 phase (Bowie et al., 2006). This
selective settlement functions as an efficient mechanism to remove aberrant HSCs.
(120) Nakamura and colleagues found that in utero exposed A-bomb survivors generally
did not carry chromosome aberrations in their lymphocytes, although they did observe asmall increase in the aberration yield at doses below 50 mGy (Ohtaki et al., 2004). This could
be the underlying mechanism for the lack of leukaemia among in utero exposed A-bomb
survivors. A similar lack of chromosome aberrations was observed for fetal irradiation inmice (Nakano et al., 2007). A recent study by the same group has indicated that the removalof in utero exposed HSCs is somewhat “leaky”, and clonal expansion of surviving HSCs canbe detected (Nakano et al., 2012). These studies can be explained by two assumptions. Firstly,
fetal HSCs do not have to be more sensitive to cell killing by radiation. Secondly, fetal HSCs
with chromosome mutations are preferentially removed during fetal to neonate stages,
possibly by competition for residency in the bone marrow niche. Although further studies aredefinitely needed, the tissue level competition is likely to serve as an effective filter toremove aberrant HSCs. A similar explanation can be made for the low sensitivity of fetal
exposures to radiation induction of intestinal tumours in the ApcMin/+ model mice, as
discussed in the previous section (Ellender et al., 2006). During fetal development, theintestine is formed as a simple tube with a layer of stem cells (Crosnier et al., 2006). In thecase of mice, the first differentiation of the villus formation takes place in day-15 fetuses.
However, crypt formation begins only on day 7 after birth. This suggests that the stem cells
settling into the crypt stem cell niche are few compared with a large number of fetal-stage
ISCs. This leads to strong competition among fetal stem cells which is likely to remove such
aberrant cells for contributing to the maintenance of the adult intestinal tissue.
(121) The competition-mediated elimination of aberrant cells during neonatal stages islikely to operate not only for radiation-damaged stem cells but also for spontaneously
aberrant cells (Nakamura, 2005). ALL is a major childhood cancer with characteristictranslocations specific to certain types of leukaemia. With the examination of cord blood
samples by the polymerase chain reaction (PCR), around 1% of newborns were found tocarry the TEL/AML1 fusion gene, one of the major translocations specific to childhood ALL.
This translocation was shown to be generated during normal fetal development (Mori et al.,2002). In fact, the number of translocation carriers is higher by two orders of magnitude than
the incidence of childhood leukaemia of this type, suggesting a possible elimination of thecells with predisposing mutations during childhood growth. The elimination of predisposing
cells after birth was suggested by the fact that the background incidence of ALL is highest ataround 3 years of age, then declines rapidly with an increase in age to 20 years, and it
increases again in the elderly (Smith, 2005). It is tempting to speculate that the stable lodging
of HSCs into the bone marrow niche during postnatal stages of development, may serve as abarrier to select out unfavourable cells with any predisposing mutations.
(122) Such elimination of cells with cancer-predisposing mutations may operate for manyother childhood cancers of sporadic types (Ries et al., 1999). Neuroblastoma, the tumour ofneural crest origin, is one of the commonest malignancies among children and its incidence,
before 15 years of age is roughly 8 in 10,000. However, necropsy samples of babies dying
within 3 months after birth demonstrated that precancerous lesions are rather common, andfound at a rate of roughly 1 in 200 (Beckwith and Perrin, 1963). Thus, the occurrence of thelesion in newborn babies and the neuroblastoma incidence differ considerably, suggestingthat most of the precancerous lesions are eliminated by some mechanisms. In accordance tothis, the incidence of tumours is highest during the first few months after birth, and declines
66

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rapidly to almost zero at an age of 15 years (Goodman et al., 1999). Similar patterns of a
rapid decline of the incidence after birth are noted for other childhood cancers such as
retinoblastoma, Wilms’ tumour and hepatoblastoma. For those tumours, terminal
differentiation of precancerous cells may be involved as a mechanism of the decline, in
addition to real elimination of aberrant/premalignant stem cells as in the case of ALL.
(123) Mouse data on fetal exposures tend to demonstrate that the risk is minimal at earlystages of gestation. However, exposure at 17 days of gestation was shown to induce cancers
of lung and pituitary gland (Sasaki, 1991). It is interesting to note that these two organs arewell developed at day 17 of mouse gestation (Yu et al., 2004; Sheng and Westphal, 1999).
Thus, even the fetal exposure is capable of inducing cancer at the perinatal stage, wherecertain tissues are well developed. In contrast to mice, humans have a much longer period of
fetal development, and therefore it is likely that certain tissues at perinatal stages are as
sensitive as after birth. Wakeford (2008) noted that 90% of the pelvimetric diagnoses in the
OSCC were performed at the last month of gestation. So, such late stages of fetal
development might exhibit a similar sensitivity to radiation carcinogenesis as that of theneonatal stage after birth. On the other hand, the remarkable similarity in the RRs of allchildhood cancer types in the study raises a concern on the causal relationship to prenatal xray examinations (ICRP, 2003; Boice and Miller, 1999). It is also noteworthy that the lifetime
RRs of childhood exposures are quite variable among cancer types (UNSCEAR, 2013).
3.7.3. High sensitivity of children to cancer induction from radiation(124) It is well recognised that children are highly sensitive to radiation induction ofleukaemia and some solid tumours (ICRP, 2003). The sensitivity of children to radiationinduced cancer has been reviewed extensively in a recent publication from UNSCEAR, andthe high sensitivity is strongly dependent on the cancer type (UNSCEAR, 2013). Children are
more sensitive for about 25% of 23 cancer types analysed when compared to adults. Thesetypes include leukaemia, thyroid, skin and brain cancer. Children have the same sensitivity as
adults for about 15% of cancer types including bladder, and less sensitivity for about 10% ofcancer types including lung. As for 20% of cancer types, the data are not sufficient for
concluding a difference in the sensitivity, and for 30% of cancer types, no increase in the risk
is observed after radiation exposure. The ERR of radiogenic cancer is inversely related to theage at exposure for many cancer types, i.e. high for the young with an age-dependent declineof the risk (Preston et al., 2007). The ERR per Gy for ALL is more than 15 for children lessthan 10 years old and the risk declines rapidly with increasing age (Hsu et al., 2013). It isnoteworthy that even with the high ERR for early onset ALL, the EAR for such cancer israther low because the background incidence of cancer is considerably lower in children thanin adults. The ERR per Gy for adult solid cancer following exposure in childhood is as highas 3-5 which declines sharply as the age at exposure increases. In addition, the latency ofcancer development is relatively short for some cancers after childhood exposures, e.g. theminimum latency interval for thyroid cancer occurred within 4 years in children after the
Chernobyl accident (Section C.1.). As a consequence, the lifetime ERR per unit dose for solidcancer after childhood exposures is around 1.0 (Pierce et al., 1996).
(125) The higher sensitivities of children for radiation induction of leukaemia and some
adult solid cancers are considered to be due to the high proliferation rate of the stem cells andprogenitor cells in children. However, these cells are also proliferating rapidly in embryos
and fetuses, and the sensitivity to radiation carcinogenesis for these stages does not appear as
sensitive as in childhood as discussed at length in the previous sections. Therefore, a cellularfeature such as proliferation alone is unlikely to explain the high sensitivity to radiation67

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induced cancer after exposure in childhood. However, there is one feature of a tissue thatdiffers considerably between children and adults, which could contribute to the highsensitivity of children to radiation carcinogenesis. As discussed, the adult stem cell niche isestablished around perinatal stages, although the time of establishment is likely to vary tissue
to tissue, and species to species. During childhood growth, the stem cell niche together with
its stem cells increases in number as a unit to match the demand of the body growth, asshown in Fig. 3.1.

Fig. 3.1. Expansion of tissue stem-cell niche multiplicity during childhood growth. (permission
needed)(126) In the case of the intestine, this process of stem cell/niche expansion isaccomplished by fission of crypts (Fujimitsu et al., 1996). The fission starts with an increasein crypt size which then splits from the bottom to form two crypts. The increased size of acrypt means an increased availability of crypt niches which eases the competition of stem
cells within a niche. In addition, competition between niches is also eased. The end result of
these is a better chance for an aberrant stem cell to remain to accumulate more mutations in
the intestine. Thus, the process of expansion of the number of stem/niche units could
potentially contribute to a higher sensitivity to children for the development of cancer afterradiation exposures.
3.7.4. Risk of carcinogenesis from exposures in adulthood(127) The risk of radiation carcinogenesis in general decreases inversely with age at
exposure. The risk also decreases by attained age after radiation exposure. Adulthood
exposures give a moderate risk as compared with childhood exposures, and this can partly be
explained by a change in the cellular features of stem cells. For example, childhood stem
cells divide frequently in a symmetrical fashion to cope with demands for the bodily growth,
while adult stem cells do so less frequently. This makes the former more prone to mutate thanthe latter. Also, the stem cells during childhood growth are expected to experience lesscompetition when the stem cell niche increases in number in response to the demand of68

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bodily growth as discussed above. Indeed, adult stem cells are under stronger competition
than stem cells of childhood growth, which is likely to keep the risk low, as discussed in
section 3.3.2.
(128) Elimination of aberrant stem cells has been demonstrated in animal models. In thecase of mouse mammary carcinogenesis, radiation exposures induce many more initiatedcells than those progressing to full malignancy (Adams et al., 1987; Kamiya et al., 1995).
Indeed, it was shown in a rat mammary carcinogenesis model that the frequency was as highas 1 in 13 irradiated mammary clonogenic cells which could not be accounted for by aspecific mutation induced by irradiation (see paragraph B55 of Annex B). Even with highly
frequent initiated cells, neoplasias arise much less frequently, indicating either efficientelimination of such cells, or suppression of their aberrant phenotypes.
(129) As for radiation exposure in adulthood, the ERR at some specified time after
exposure is generally smaller than that for childhood exposures. The risk rises within a few
years for leukaemia and after 10 years or more for solid cancer, and the elevated ERR
eventually starts to decline with an increasing attained age of those exposed. This pattern ofelevation and decline of cancer risk has been observed repeatedly, but the most reliable data
come from epidemiological studies (Boice et al., 1985) and notably of A-bomb survivors(Preston et al., 2007; Richardson, 2009; Hsu et al., 2013). Based on the multistage
carcinogenesis model by Armitage and Doll (1954), the RR of a population given one hit byacute irradiation was predicted to decline over the attained age by a rate of 1/age (Pierce and
Mendelsohn, 1999). The rates of decline of RR were estimated in the recent compilation of
the A-bomb survivor data. Although the estimates varied among cancer types, the rates werein general in the range of around 1/age2 (Preston et al., 2007). In addition, a study of theradon exposed uranium miners indicated that the RR of lung cancer declined about 50% foreach 10 years after they stopped working in mines, suggesting the decline to be proportional
to around 1/age3 (Tomasek et al., 2008). These rates of the RR decline are higher than 1/age
and may suggest the loss of initiated/precancerous cells in a tissue over time, as discussed
earlier.
(130) Overall, the above-described age-dependent sensitivities to radiation carcinogenesis
can be summarised as follows: embryo and fetal stages to be low to moderate, children to behigh and adult to be low. However, mechanistic insight for this pattern of radiosensitivity is
still lacking. In the past, it was naively assumed that the high sensitivity to radiation
carcinogenesis for children could be attributed to high rates of proliferation of somatic cells at
this stage of human life. However, this simple assumption makes it difficult to explain why
the fetal stages with even higher rates of proliferation are not associated in general withextremely high sensitivity to radiation carcinogenesis, although the latter remains somewhat
controversial.

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3.8. Summary

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• There is evidence for a hierarchical-type cell renewal lineage and stem cell niche in all theanimal tissues considered in detail in this report. Most evidence pertains to haematopoiesis,
GI tract and epidermis, which have greater renewal rates than in mammary tissue, lung,thyroid and bone.
• The target cells for carcinogenesis continue to be considered primarily the tissue stem cells.
In haematopoietic tissue and colonic mucosa, there is evidence of an age structure within
the stem cell population. In haematopoietic tissue, colonic mucosa and epidermis, there issome evidence for progenitor cells being target cells as well. The microenvironmental

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69

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•

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•

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•

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•

•

•

niche is an important regulator of stem cell maintenance and a modifier of stem cell
response.
The immortal DNA-strand hypothesis was proposed which would protect stem cells from
replication-mediated mutation. There is evidence in support of such a mechanism in small
intestinal crypts, mammary epithelium, some muscle satellite cells and progenitor cells,
and some CNS cells. It was also inferred from studies of tongue epithelium. However, it
was found not to apply in HSCs. Since it is not a universal phenomenon, the relevance of
this highly interesting hypothesis is yet to be proven.
The radiation-induced incidence of cancer for different organs and its associated tissue
weighting factors relate to carcinogenesis of target cells. Carcinogenesis depends primarilyon three mechanistic factors: (1) the number and sensitivity of stem cells to radiationinduced mutation; (2) the retention of mutated stem cells in a tissue; and (3) the population
size of stem cells with a sufficient number of predisposing mutations. It is postulated inthis report that the ERR function reflects largely the cellular sensitivity to radiationinduced mutation and the retention of predisposed stem cells. The EAR function reflects inaddition to those two factors, the population size of the predisposed stem cells, thus being
more complex but comprehensive in relation to radiation carcinogenesis of stem cells andstem cell populations. At present, there is a lack of definitive evidence for one or more ofthese factors contributing to radiation risk for the tissues considered in this report.
An LQ model describes fairly well the dose-response relationships for tumour induction in
animal systems, and values of the DDREF are generally in the range 2 to 10 (UNSCEAR,
1993; NCRP, 2005), i.e. generally a higher range than that proposed for human application.
With dose fractionation and protraction to 25 days, the results for mammary tissue andlung are consistent with LQ predictions, and for thyroid, the low-dose-rate 131I and lowdose acute x-ray data are consistent. For AML, further protraction reduces tumour yield,
whereas for skin cancer further protraction increases the yield. Hence, protraction effects
are clearly tissue dependent. The stem cell competition model, where damaged stem cells
are outcompeted by undamaged stem cells for residence in the stem cell niche, istheoretical but with some supporting evidence which might help explain the different risks
among tissues from chronic irradiation scenarios.
The LNT model continues to form the foundation of the ICRP risk assessment for low
dose and low dose rate exposures. For tissues where transfer of risk is based mainly on
ERR, the LNT model in combination with the RR model implies that any risk-reducing
actions which decrease the underlying background risk of some cancer types also might beeffective in reducing the risk of radiation-related cancer. It is, however, clear that the LNTmodel is a tool for radiation protection purposes to assess the risk of all tumour types
combined and does not apply for some cancer types such as leukaemia and skin cancers.
Furthermore, it is not applicable to those cancers not consistently increased following
radiation exposures, such as the rectum, small intestine, pancreas, uterus, prostate andkidney parenchyma (Ozasa et al., 2012) as well as the testis, cervix and lymphomas.
In addition to the quality control systems of DNA repair at the molecular level andapoptosis at the cellular level, stem cell competition is likely to function as a quality
control at the tissue level which could adequately explain the age dependence of radiation
carcinogenesis. In addition, stem cell competition offers a new mechanism for the sparingof risks of stochastic effects at extremely low dose rates such as a few mSv per year.
Age-dependent sensitivities to radiation carcinogenesis can be summarised as follows:
embryo and fetal stages to be low to moderate, children to be high and adult to be low,70

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with possibly higher sensitivity as people age further. However, mechanistic insight forthis pattern of radiosensitivity is still lacking at present.

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3.9. Recommendations for future research

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1. Knowledge of the stem-cell/niche systems and their sensitivity to radiation injury resulting
in carcinogenesis in bone marrow, large intestine and skin is much more developed thanfor other tissues at risk such as mammary gland, thyroid, lung and bone. Hence there is aneed to further investigate the stem cell systems in these latter more-slowly renewing
tissues, their controlling factors, and their mutational/mechanistic responses to acute andprotracted irradiation.
2. The immortal-DNA-strand hypothesis, whereby the parental DNA template is retained
during asymmetric stem-cell divisions so that the mutational burden in the stem-cell
population is kept low, might act to protect against radiation carcinogenesis. There isevidence in support of this mechanism in some tissues considered in the present report (e.g.
small-intestinal crypts, mammary epithelium, and epidermis), but the mechanism wasfound not to apply in HSCs. Hence it would be informative to study further tissue types,
and to attempt to measure purportedly-relevant carcinogenic mutations in stem cells
following acute or protracted irradiations in tissues which show this phenomenon.
3. The concept of competition between normal and radiation-injured stem cells for residence
in the stem-cell niche, which might act to give less carcinogenic events than expected after
irradiation, is supported by studies in haematopoietic tissue after acute doses of 1 Gy or
more. It would be useful to study this effect after lower doses and in particular after low
chronic-radiation exposures, as well as in other tissues where possible.
4. The numerical value of the DDREF appears to be lower from recent evidence in somehuman populations than in experimental animal systems, and the reasons for this are
currently unclear. Also, regarding the effect of age-at-exposure, there are inconsistencies
between the evidence in human populations and in experimental animal systems regarding
carcinogenesis at the fetal and neonatal stages of development. Reconciling thesedifferences at the biological level should help in underpinning the consistency androbustness of the protection system.

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ANNEX A: HAEMATOPOIETIC TISSUES: ROLE PLAYED BY STEM CELLS AND
LINEAGE-COMMITTED PROGENITOR CELLS IN RADIATION-INDUCEDLEUKAEMIA

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A.1. Radiation-induced leukaemias and lymphomas

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(A1) Leukaemias and related neoplasias of the blood-forming system of body areprominent, late-arising pathologies of prior, sufficiently damaging radiation exposures. As ageneral class of radiation-induced “cancer”, leukaemia is the earliest arising type thatmanifests following exposure, and is arguably the best defined, most radiogenic form ofcancer. Induction of leukaemia by ionising radiation is well documented in both humans andin animals (ICRP, 2005, 2012; UNSCEAR, 2000, 2006; 1990; 2006).
A.1.1. Epidemiological studies(A2) As a single disease entity, radiogenic leukaemia has an appreciably higher ERR thanany of the other late-arising pathologies that have manifest within the A-bomb survivor
cohort. As shown in Table A.1., the estimated ERR per Gy of exposure for leukaemia (inaggregate for both sexes, using a linear dose model) within the A-bomb (LSS) survivor
cohort is approximately 8-10 fold higher than it is for the solid cancers, and about 30-35 foldhigher than for the non-cancer diseases. The latter category selectively excludes a group of
non-neoplastic blood disorders, e.g. the myelodysplastic syndromes (MDS), which havepathological relationships with evolving leukaemia and in turn have comparably elevated
ERRs to that noted for the radiogenic leukaemias (Iwanaga et al., 2011), although the time
pattern is peculiar in that increased risk are high some 40 years after exposures and does notseem to decline as is the case for radiogenic leukaemia over time. MDS was not a diseaseclassification until the 1980s, so that early occurrence could not be effectively evalued.
Gender differences have been observed, albeit relatively small: for males and females, theestimated ERRs for leukaemia mortality have been estimated to be respectively 4.6 (95% CI:
3.0, 6.9) and 3.9 (2.5, 6.1) per Gy, based on a linear dose-effect model (Ozasa et al., 2012).
Estimates of absolute rates (i.e. EARs), by contrast, tend to indicate greater differences
between the sexes: e.g. BEIR VII (2006) reported an EAR for leukaemic deaths in males tobe 1.62 per 104 PY-Sv, whereas for females the estimate was considerably lower, 0.93 per
104 PY-Sv. Additional estimates of leukaemic risk based on alternative radiation-exposedcohorts have been developed and reported, and many indicate generally comparable levels of
elevated risk, but with a much greater level of uncertainty; e.g. an ERR/Gy estimate of 1.93,
with 95% CI (<0, 8.47), was reported for a massive cohort of occupationally exposed nuclear
workers (~400,000) from 15 countries (Cardis et al., 2007). The lack of statisticalsignificance in such large studies indicates that chance cannot be discounted as one possible
explanation for the observed association, related in part to the relatively low dose and narrowdose range of the exposed population.
(A3) The risk of developing radiogenic leukaemia is related to the extent of radiation
exposure. Within the LSS cohort of A-bomb survivors, at very low doses ranging from 0.005
to 0.1 Gy, an estimated 5% of the leukaemias likely developed as a result of the irradiation.
At higher exposure levels of 0.1 to 0.5 Gy, an estimated 36% of the cases were radiation
associated. At a still higher range of doses of 0.5-1.0 Gy, 66% of the cases were estimated to
be radiation related, and at or above 1.0 Gy, it was 86% (Table A.2.).
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Table A.1. Estimated ERR per Gy for major causes of death within the A-bomb (LSS)
surviving cohort 1950 to 20031
Cause of death

GenderNumber2Model

P-value

Est ERR/Gy3,4

95% CIAll causes
Leukaemia
Leukaemia““
Leukaemia5
AML Leukaemia5
CML leukaemia5
ALL leukaemia5
MDS6
Lymphoma
Multiple myeloma
Solid cancers
Non-cancer
diseases7.8
Blood diseases

M&F
M&F
M&F
M
F
m/f
M&F
M&F
M&F
m/f
M&F
m/f
m/f
m/f

50,620
318
318
163
155
310
124
58
19
47
284
93
10929
25618

L
L/Q at 1Gy
L/Q at 0.1Gy
L
L
L
L
L
L
L
L
L
L
L

<0.001<0.001<0.001<0.001<0.001<0.001>0.05>0.05< 0.001< 0.001

0.22
3.1
0.15
4.6*
3.9*
4.7
4.3
6.4
3.7
4.3
0.16
0.54
0.47
0.13

0.18,0.26
1.8,4.3-0.01,0.31
3.0.6.9
2.5,6.1
3.5,6.4
2.7,6.6
3.0,13.7
0.8,13.0
1.6,9.5-0.13, 0.59
-0.04, 1.58
0.38,0.56
0.08,0.18

m/f

238

L

<0.0011.7

0.96,2.7

1

Data obtained from Ozasa et al. 2012 (except where otherwise indicated).
Numbers listed from the LSS cohort registry (~120,000 individuals).
3
Estimates for ERR values are sex-averaged (* indicates ERR estimate is for a single sex) and basedon a linear dose model for all causes of death (all categories), except for the ‘leukaemia’ value
which is based on a LQ model and for an exposure of 1 Gy (at a lower 0.1 Gy exposure, the ERR
estimate is 0.15 with 95% CI of -0.01 and 0.31).
4
ERR estimates for leukaemia, based on a linear dose model, yields higher ERRs for both males(4.6/Gy with 95% CI: 3.0, 6.9) and females (3.9/Gy with 95% CI: 2.5, 6.1).
5
ERR estimates (without effect modification), along with 90% CI, are listed for both the aggregate(sex-combined) leukaemia-related mortality response, as well as for the leukaemia mortality
responses of major subtypes of the radiogenic leukaemias, as listed in Richardson et al. (2009).
6
MDS, Myelodysplastic syndrome: a preclinical haematological disease that often precedes the onsetof myeloid leukaemia. Data presented are from Iwanaga et al. (2011): 47 MDS patients within the
LSS cohort of 22,245 Nagasaki subjects.
7
ERR estimate (excluding non-neoplastic blood diseases) made using a linear dose-effect model, andbased on the aggregate of non-cancer deaths recorded from 1966-2003.
8
Non-neoplastic blood diseases that were excluded from the non-cancer disease category.
2(A4) The above updated estimates closely correspond to those reported earlier (Pierce etal., 1996; Preston et al., 1994), when approximately 44% of the total leukaemic deaths (78 of
176) observed in the LSS cohort for doses above 0.005 Sv were attributable to the A-bomb
irradiation.
(A5) Points to note are: (1) estimates clearly indicate a rising proportion of exposurerelated leukaemias related to dose; (2) the dose-dependent rise is not simply linear, but has
upward curvature (Fig. A.1.) which can be fitted using an LQ function; (3) estimates andmodels pertain to exposure levels ranging from ~0.005 Gy to 3.0 Gy; and (4) specific types ofleukaemia (e.g. acute, chronic, myeloid, lymphocytic, etc) give rise to different risk estimates.
This is illustrated in Table A.3 by the marked variance in the excess number of leukaemic
deaths resulting from spinal irradiation for ankylosing spondylitis (Darby et al., 1987; 1990).
Note that in the Darby et al. study, the “observed to expected ratio” of leukaemic cases was73

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calculated to be ~3.17, based on the 39 leukaemic deaths observed, and 12.29 which had been
expected within a cohort of 14,106 patients and a follow up period of ~48 years. An earlier
study of the same cohort by Smith and Doll (1982) reported that these patients had averagebone marrow doses ~3.21 Gy and a calculated ERR of 0.98 per Gy. Also, note that a more
recent follow-up study on this same cohort was report by Weiss et al. 1995: in contrast to
earlier reports, the ratio of ‘observed-to-expected’ cases of leukaemia, excluding CLL, was~11 during a period 1-5 years following initial radiation treatment; this ratio declined to ~1.9
for the 25-year period following initial exposure; the ERR for ALL, a leukaemia subtype, wasestimated to be ~12/Gy using an LQ dose model and an average spinal marrow radiation doseof ~4 Gy.
Table A.2. Leukaemic mortality 1950-2000 within the A-bomb LSS cohort of survivors1
Marrowdose (Gy)

Subjects

Person-years

Cases

Estimated
excessEstimated
attributable risk (%) 2<0.0050.005-0.1
0.1-0.2
0.2-0.5

37,40730,387
5,841
6,304

1,376,521
1,125,891
208,445
231,149

92
69
14
27

0.1
4.0
4.7
10.4

<0.2
5.6
33.6
38.5

0.5-1.0
1.0-2.0
2.0+
Total

3,963
1,972
737
86,611

144,276
71,485
26,5893,184,356

30
39
25
296

18.9
27.7
28.2
93

63.0
71.0
>100
31.4

1
2

From Preston et al. Radiat Res 162: 377-389, 2004, with added ‘attributable risks’.
Estimates of ‘attributable risk’ for leukaemia added to the Preston et al. table by the present
authors.

Leukemia
35

Excess leukemic cases

30
25
20
15
10
5

Excess leukemic cases

0Linear model
L/Q model-5<0.0050.005-0.1 0.1- 0.2

0.2- 0.5

0.5- 1.0

1.0- 2.0> 2.0

Marrow dose (Sv)

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Fig. A.1. Linear-quadratic (LQ)-fitted radiation exposure versus leukaemic incidence relationship.
Excess number of total leukaemic cases recorded within the LSS cohort of the A-bomb survivors.
Note the marked and significant (P=0.002) upward curvature of the response at the higher doses(graph drawn from tabulated data of Preston et al. 2004). (Permission needed)

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Table A.3. Excess number of leukaemic deaths resulting from spinal irradiation forankylosing spondylitis1, 2
Leukaemic subtype

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ObservednumberNumber of deaths
Expected
Ratio ofnumberobserved/expected

Myeloid leukaemia:
Acute
17
4.34
3.92
Chronic
3
2.05
1.46
Unspecified
4
0.71
5.63
all subtypes
24
7.10
3.38
Lymphocytic leukaemia:
Acute*
20.93
2.15
Chronic2
2.38
0.84
Unspecified
3
0.38
7.89
all types
7
3.69
1.89
Unspecified leukaemia:
3
0.28
10.71
all types
36
11.29
3.198*
1
Darby et al.1987, reported in BEIR V 1990.
2
Patients received fractionated regimens of partial-body, x irradiation of the spine; average,
cumulative exposures to the bone marrow were estimated to be 3.21 Gy (Smith and Doll 1982).
* Follow-up study by Weiss et al. (1995) reported on 60 cases of leukaemia among 13,914 patientswith ankylosing spondylitis and treated by fractionated radiotherapy: (a) average radiation dose tobone marrow was estimated at 4.38 Gy; (b) observed-to-expected ratios for periods 1-5 years and1-25 years post initial treatments were ~11 and 1.9 respectively; and (c) using an LQ dose model,
the ERR at 1 Gy exposure for ALL was estimated at ~7/Gy.

(A6) Estimates of leukaemic risk vary depending on both (a) the subject’s ‘age’ at the timeof radiation exposure and (b) the subject’s ‘attained age’ following exposure (Preston et al.,2004). For the very young (0-9 years), the EAR estimate is 0.66 per 10,000 PY Sv (90% CI:
0.13, 1.3) and with a negative, ‘time-since-exposure’ power function of -1.1 (-1.6, -0.07); foryounger adults, 20-39 years of years, the EAR estimate is elevated at 1.3 (0.3, 2.5) and has asmall, but positive, ‘time-since exposure’ power function of 0.03 (-0.6, 0.7); older adults (≥40
years), the EAR estimate is further elevated at 1.9 (0.4, 3.9), with a relatively strong, positive‘time-since-exposure’ power function of 0.5 (-0.3, 1.3). Estimates of ERRs vary markedly
according to ‘age’ as well: a temporal ‘wave’ of markedly elevated risk estimates (e.g. ERR~10-100/Gy) has been noted in very young exposed cohorts over the initial decade or two
post-exposure (Little, 2009; Richardson D, 2009; Wakeford, 2008, 2012; Weiss et al., 1995).
The most recent analysis of leukaemia among A-bomb survivors, however, has not shown aslarge a risk as seen in these previously analyses (Hsu et al., 2013). As the age of the exposedcohort increases (i.e. both the age-at-exposure, as well as the attained age at the time ofanalyses), the magnitude of the RR declines appreciably, but still remains significant(Kodama et al., 2012).
(A7) Risk estimates for leukaemia associated with in utero radiations have varied widely:for example, markedly elevated and statistically significant estimates (ERR ~50/Gy) ofchildhood leukaemia associated with antenatal diagnostic radiography continue to be reported
from updated case-control studies of the OSCC (Wakeford, 2008); whereas, by contrast,
significantly increased leukaemic risks have not been demonstrated in any cohort study of inutero irradiated individuals (NCRP Report 174, 2014), e.g. offspring of Mayak female
nuclear workers (Schonfeld et al., 2012); children of Japanese A-bomb survivors (Jablon and75

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Kato, 1970), or 40,000 prenatally exposed children in the UK followed prospectively (Court
Brown et al., 1960).
A.1.2. Experimental animal studies(A8) The results from experimental animal studies are generally consistent with the humanepidemiological observations and associated risk estimates. Although the dose-dependent
nature of induced leukaemia from both acute and fractionated/chronic whole-body ionisingradiation has been demonstrated in a wide variety of experimental animal species under
different experimental conditions, the relationship between exposure dose and leukaemic
incidence is: (a) quite variable, depending on the nature of the induced leukaemic subtype;(b) dependent on substantial levels of bone marrow exposure and damage; and (c) tends not
to conform to a simple, LNT function (1990). Experimental studies have not found consistentevidence that in utero exposure to radiation increases the risk of leukaemia in offspring(NCRP Report 174, 2014; Upton 1960).
(A9) Of the experimental radiation-induced lymphohaematopoietic malignancies, thethymus-associated T-cell lymphomas have been most studied: for instance, in select strains of
mice, these T-cell lymphomas can be readily and reproducibility induced at extremely highfrequency by minimal, fractionated dose regimens (e.g. 4 x 2 Gy fractions delivered at
weekly intervals for a month) (Humblet et al., 1997; Utsuyama and Hirokawa, 2003). Withuse of the latter model, exposure thresholds can be readily demonstrated, as well as theprotective actions of bone marrow shielding or infusions of normal, unirradiated marrow(Humblet et al., 1989; Kaplan, 1974; Sado et al., 1991). Age of the animal and of the thymus
itself plays a critical role following such lymphoma/leukaemic-inducing regimens of ionisingradiation; i.e. malignant process(es) are strongly influenced by age-related factors of thethymic microenvironment, the bone marrow and the host environment (Utsuyama andHirokawa, 2003). Early work suggested that these thymus-associated T-cell lymphomas (aswell as radiation-induced myeloid leukaemias) in experimental mice were largely the resultof radiation-induced activation of latent leukaemia retrovirus (Gross, 1959; Janowski M. etal., 1987). However, it is now recognised that in a number of strains of mice (e.g. NFS,
C57BL/6 mice) with well characterised susceptibilities to radiation-induced thymic
lymphomas, exposure-related malignancies are manifested that are largely free oftransforming endogenous ecotropic retrovirus (Ihle et al., 1976; Kominami and Niwa, 2006;
Okumoto et al., 1990).
(A10) Molecular/cytogenetic analyses of radiation-induced leukaemias in a number ofmouse strains have revealed a number of predisposing genetic lesions, e.g. an allelic loss on
chromosome 4 of the LYrs/TLSR5 locus in >95% of pre-B lympho-myeloid leukaemias ofirradiated CBA/H mice (Cleary et al., 2001).
(A11) Experimental myeloid leukaemia has been somewhat less studied than thelymphomas or lymphocytic leukaemias. The nature and patterns of induction of myeloid
leukaemia seem to align more closely to the noted disease induction patterns in humans. For
example, in select strains of myeloid leukaemia-susceptible mice (CBA, RFM, C3H), theradiation exposure versus myeloid leukaemia incidence curve rises proportionately withincreasing doses of acute, whole-body γ or x irradiation (Major and Mole, 1978; Mole et al.,
1983; Otsu et al., 1995; Upton, 1985). The response at very low levels of exposure (frombackground levels to approximately 1 Gy) is fairly linear. At doses from 1 to 2-3 Gy, theincidence curve tends to exhibit upward curvature; and at doses greater than 3 Gy, the curve
tends to bend over downwards.

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A.2. Relevant data for various radiations and exposure types
A.2.1. Epidemiological data
(A12) Although radiobiological relationships concerning protracted, low-dose/dose-rate
exposures are clearly less well-defined in humans compared to experimental animals, it
seems highly likely that basic relationships are comparable and that further, detailed
epidemiological work will show this to be the case. In support of this contention, BEIR-VII(BEIR VII, 2006) ERR estimates for leukaemia (excluding CLL) within select, chronically
exposed ‘worker populations’ were significantly lower than those reported for A-bomb
survivors (i.e. an ERR of 2.2/Gy for ‘all ages’ of ‘workers’ chronically exposed to low dosesof radiation is significantly lower than the earlier reported ERR value of 4.2/Gy for A-bomb
survivors who were acutely exposed to relatively high doses of low-LET radiation).
(A13) Epidemiological studies of leukaemia incidence within the chronically exposed
Techa river cohort, however, have indicated a strong, dose-dependent response with ERR
values of 4.2 and 4.9 for all subclasses of leukaemias, except CLL (Krestinina et al., 2010 ;Krestinina et al., 2005 ). These elevated ERR values might be due to the nature of the chronic
exposures: namely either mixed external/internal exposures of both high and low-LET
radiation, or perhaps due to variable rates of exposure over time. If the latter is correct, thenthe elevated ERR values might be more consistent with prevailing radiobiology theory (i.e.
little to no sparing of cancer/leukaemia incidence with protraction of low dose/dose-rate,high-LET type exposures).
(A14) The overall leukaemia-inducing potential of internally-deposited radionuclides,
especially bone-seeking, α-emitting nuclides (e.g. in 226Ra/228Rn exposed dial painters (Spierset al., 1983) and in 239Pu exposed nuclear workers at the Russian Mayak nuclear complex(WHO, 2001), is somewhat less than what one might expect by comparison to theleukaemogenic efficiencies of external, whole-body exposures with low-LET x- or γ-rays.
This is despite accounting for estimated dose-equivalents to tissue targets such as theendosteal layer at the bone/marrow interface, and the substantial differences in LET ofvarious types of radiations. There is no question that internally-deposited radionuclides havethe potential to cause leukaemia in humans: this has been amply documented under a variety
of exposure conditions and with various radionuclides (Andersson et al., 1993; Wick et al.,2008). The difference seems to lie in terms of the leukaemogenic efficiency of the different
radiation qualities.
A.2.2. Experimentally-based animal data
(A15) The effect of both radiation quality and radiation dose rate can been seen readily by
the change in slope of the myeloid leukaemia incidence curve between total doses ranging
from background levels to doses of 1-3 Gy. Experimentally (in various strains of mice; e.g.
RFM and CBA), myeloid leukaemia incidence per Gy markedly declines with increasing
dose fractionation (or continuous exposures at very low dose rates) (Mole and Major, 1983;
Otsu et al., 1995; Upton et al., 1970). From such studies, DDREF values have been estimatedand range from 3-9 (Otsu et al., 1995). These estimates are comparable to those DDREF
estimates (i.e. 2-10) made for humans exposed chronically to low doses/dose-rates of lowLET radiation (NCRP-64, 1980).
(A16) The dose-fractionation effect on myeloid leukaemia incidence is considerably more
pronounced with low-LET x- or γ−ray exposures than with high-LET neutrons (Upton, 1985;
Upton et al., 1970). The latter LET/dose-rate effect on myeloid leukaemia incidence has a
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very significant effect on the estimated RBE values for neutrons, and probably for other highLET irradiations, For example, the estimated RBE for neutrons at high dose-rate is in therange of 2-3 for myeloid leukaemia incidence, while at very low dose rates (~ 0.01 Gy min-1)
the RBE dramatically increases to 16 or more (Ullrich and Preston, 1987; Upton et al., 1970).
Nevertheless, the latter values are much less relative to the RBE estimates of 20-50 asreported for the induction of solid tumours of neutron-exposed rodents. Regardless, thesemarkedly and significantly elevated values of RBE at low doses/dose-rates of high LET
fission-neutron exposures clearly have the potential to impact leukaemia/cancer risk
assessment process(es) (BEIR VII, 2006).
(A17) The leukaemogenic potential of internally deposited radionuclides has been verified
using various animal models under a variety of treatment protocols (Boecker, 1995;
Humphreys et al., 1987).
(A18) Short-range α-particle emitters have been used to help identify the location of thetarget cells for radiation-induced leukaemia and osteosarcoma. After radionuclide injection in
mice, cumulative radiation dose to different regions of the bone and marrow was assessed indetail from sequential microradiographs taken over a period of 15 months (Lord et al., 2001).
The relative incidence of osteosarcoma after uptake and redistribution of 239Pu, 241Am, or
233
U correlated preferentially with the dose to target cells averaged over just 0-40 µm fromthe bone surface, and for AML risk the correlation was closest for target cells in the central
sinusoidal region. Although there are caveats to this, such as cell migration during thedosimetry period, the results support the general contention of the importance of theendosteal and sinusoidal regions in radiation carcinogenesis.

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A.3. General features of haematopoietic tissues

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(A19) The haematopoietic system is an essential organ system whose primary activity is
manufacture of various species of mature, functional cells which circulate in the blood. These
blood cells include: erythrocytes for oxygen transport; leukocytes for immune defence; andplatelets for haemostasis. The cell producing capacity and fidelity are great: e.g.
approximately 2.5 x 109 erythrocytes, ~2.5 x 109 platelets, and ~10 x 109 granulocytes per kg
body weight are produced on a daily basis in humans (Williams et al., 1990).
(A20) Haematopoietic tissue is a hierarchical, self-renewing, and cell amplifying tissue,
maintained by small numbers of stem cells and early progenitor cells, whose primaryfunctions respectively are to asymmetrically self-renew following infrequent divisional
cycles and to commit to differentiate into specific blood cell lineages. The most primitive
HSCs (marrow repopulating HSCs) of humans, in a steady state, are thought to undergo 50 ormore divisional cycles during an average lifespan, producing upward of ~1015 blood cells(Kay, 1965). Note that reported estimates for replicative capacity can vary widely, ranging
from ~5 to ~1000 divisions per lifetime, depending on, but not limited to, such factors as thenature of HSC population/subpopulation and physiological state of the marrow at the time of
HSC isolation and, of course, the species from which the marrow was derived. To illustrate
the later, small subpopulations of largely-quiescent HSCs in steady-state marrow of humans
and mice replicate at significantly different rates, namely ~280 days and 145 days,
respectively; therefore, suggesting markedly different numbers of HSC replications per
lifespan for individuals within these species (Catlin et al., 2011; Kay, 1965; Harrison, 1979a,b; Pietras et al., 2011; Shepherd et al., 2004; Shepherd et al., 2007). These stem cells do not
function independently, but rather in a well-orchestrated manner dictated largely by stromal
cell niches and associated endogenous signalling networks (Fig. A.2.) (NCRP-150, 2005).

78

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Progenitors

Hematopoietins

Endoregulatory
processes

Exoregulatory
processes

Self-­‐renewal

Stromavs.
Differentiation

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Fig. A.2. Interrelationships of bone marrow stroma, haematopoietins, and haematopoietic progenitor
cells. (Figure reproduced courtesy of the NCRP) (permission needed)(A21) The most primitive of the HSCs is exceedingly rare cell types, occurring at estimatedfrequencies from 10-4 to 10-5. The current consensus is that there are two major HSC
compartments within the marrow. The first, often labelled the Lt-HSC compartment, is moreprimitive, quiescent by nature. They are responsible for the long-term renewal andmaintenance of haematopoiesis. The second, labelled the St-HSC compartment, somewhat
quiescent, with higher self-renewal, and shorter term responsibilities for repopulatingprecursor marrow niches (Yin and Li, 2006). Under steady-state conditions, these cells arelargely quiescent (>95% in a non-cycling G0 state), but when forced into a divisional cycle
they preferentially self-renew. These HSCs reside within three domains: (i) endosteal; (ii)parenchyma cord; and (iii) vascular sinus regions (Shiozawa and Taichman, 2012; Fig. A.3).
Through an ill-defined process, selected daughters of the stem cells take alternative steps andbegin to commit to one specific lineage, thereby losing some self-renewing capacity but
gaining proliferative activity. Through successive divisional cycles, sufficient numbers of
lineage-committed progenitor cells are produced in order to satisfy the production
requirements for specific cell lineages, and at this stage, proliferation stops and terminal
maturation processes begin. Count estimates for a number of these marrow compartments arelisted in Fig. A.4.

79

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Major	
  compartments

Proliferative,	
  lineage-­‐committed	
  	
  daughters	
  

Stem	
  cells &	
  early	
  progenitors
Lineage-­‐restricted	
  Repopulating	
  Non-­‐proliferating,	
  Maturing	
  cells	
  &	
  reserves

Granulocytic	
  series

Neutrophilic granulocytes

Monocytic seriesMultipotential

Erythrocytic series

. ...
. .
. . . ..

Monocytes

Eosinophilic granulocytes
Basophilic	
  granulocytes
Erythrocytes

Megakaryocytic	
  series
Femoral	  bone	  Megakaryocytes&	  Platelets

Lymphocytic	
  seriesT-­‐lymphocytes...
.

B-­‐lymphocytes..

NK-­‐lymphocytes

Vascular	
  sinuses
Parenchyma/cords

Major	
  stemcell	
  domains
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Endosteal region

osteoblasts
stromal cells
endosteal HSCs
endosteal lining	
  cellsosteocytes

osteoclasts

stromal/reticular	
  cells

myelocytic cells
adipocyte/fat	
  cellssinus	
  HSCsparenchyma	  HSCsFig. A.3. General features of haematopoietic tissues, modified from an NIH online report (NIH Report,
2008). (Bone cross-section drawing reproduced with the courtesy and permission of Terese Winslow
LLC, Alexandria, VA). (permission needed)

80DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE

General	
  features	
  of	
  hematopoietic	
  tissues:Count	
  estimates1	
  Major	
  compartments1)	
  Stem	  cell	  &	
  early	
  progenitors2)	
  Proliferative,
lineage-­‐committed
daughters	
  3)	
  Non-­‐proliferating,	
  maturing	
  cells,	
  andassociated	
  reserves.
Major	
  stem	  celldomains1)	
  Endosteal region2)	
  Paraenchyma/cords3)	
  Vascular	
  sinsuses4)	
  Blood	
  (total)	
  

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HSCs	
  (total)	
  =	
  0.84-­‐1.26	
  x	
  108	   (0.007-­‐0.01%)-­‐ Lt	  HSCs	  (repop.)	
  =	
  0.13	
  x	
  108 (0.001%)-­‐St	
  HSCs	  (repop.)	
  =	
  0.71-­‐1.13	
  x	
  108 (0.006-­‐0.009%)-­‐ CD34+CD38-­‐ HSCs	
  =	
  0.13-­‐1.6	
  x	
  108	  (0.001-­‐0.013%)	
  Total	  (active)	
  marrow	
  =	
  1.26	
  x	
  1012 cells	
  All	
  lineages	
   =	
  1.26	
  x	
  1012 cells	
  (100%)-­‐Myeloid	
  =	
  0.72	
  x	
  1012 cells	
  (54.4%)-­‐Myeloblasts =	
  0.01x1012 (0.8%)-­‐ Monocytes =	
  0.018x1012 (1.4%)	
  -­‐ Megakaryocytes =	
  0.0025x1012 (0.2%)-­‐Erythroid =	  	
  0.24	
  x	
  1012 (19.1%)-­‐ Lymphoid	
  =	
  0.13	
  x	
  1012	
  (10.4%)-­‐ CD34+ cells	
  =	
  0.0126	
  x	
  1012	
   (1%)HSCs	
  (total)	
  =	
  7.4-­‐20.7	
  x	
  104
-­‐CD34+	
  (total)	
  =	
  0.52x108-­‐ HPCs	  (CFU-­‐mix)	
  =	
  0.017x108
1	
  count	
  estimates	
   based	
   on	
  average	
   man,	
  ~70	  kg	  in	
  weight.	
  Fig. A.4. Estimates of cell numbers for various major haematopoietic tissue compartments.
(permission needed)(A22) The effect of collateral GI injury and selected types of microbial flora (e.g.
Helicobacter sp.) on leukaemic progression is fairly well recognised. In contrast toconventionally-reared laboratory mice, gnotobiotic (germ-free) RFM mice appear highly
resistant to radiation-induced leukaemia (Walburg et al., 1965). In contrast, humans withvarious types of chronic infections that generate genome-modifying free radicals, appear
susceptible to leukaemia and related lympho-haematopoietic disorders (Karaoglu et al., 2004;
Maeda, 1998; zur Hausen, 2009). The precise mechanism(s) by which chronic infection andionising radiation might synergise relative to stem cell transformation remains speculative.
A.3.1. Turnover rate
(A23) There are at least 8 blood cell types that circulate within the blood, each having
defined turnover rates, distinct cell origins and lineages, with separate reproductive andmaturational histories. As such, “turnover” (as defined by “cell replacement”) of
haematopoietic tissue needs to be considered in the light of its multilineage nature. As
indicated earlier, the origin of these multiple, blood-forming cell lineages lies within theprogenitor cell compartments of the bone marrow. These small but vital cell compartments
contain a continuum of very primitive and less-primitive progenitor cells having a full rangeof self-renewal potentials and reproductive capacities, as well as varying cell cycling
activities and pre-programmed commitments for lineage differentiation. It remains uncertain,
however, whether or not the most primitive of these progenitor marrow compartments everrenew completely during the lifespan of the individual. Nevertheless it is the lifespan and81

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change in number of circulating, functionally mature cells in the blood that ultimately dictate
the turnover of haematopoietic tissue.
(A24) Under steady-state conditions, turnover rates of the different types of blood cells
within the circulation can differ by orders of magnitude: for example, the circulating pool oferythrocytes in man turns over completely every 120 days or so, while circulating pools ofblood platelets and granulocytes are replaced approximately every 5-9 days and 12-24 hours,
respectively. Under a variety of abnormal haematological conditions, these turnover rates canbe altered appreciably. As such, the trilineal haematopoietic tissues of the marrow (i.e.,
myelopoietic, erythropoietic, and megakaryocytopoietic elements) need to be both flexible
and robust, not only on a daily basis, but also throughout life. In terms of erythropoietic
capacity, approximately 1.75 x 1011 fully functional, fully mature red cells (per average ~70
kg person) are produced in the marrow and released into the blood on a daily basis in order toreplenish the daily loss of approximately 0.8% of the circulating red cell pool.
Megakaryocytic elements of the marrow need to produce and release a comparable number of
1.75 x 1011 platelets in order to make up for the daily loss of ~16% of the circulating platelet
pool. Also, the granulocytopoietic elements of the marrow need to produce daily ~7 x 1011
blood granulocytes in order to completely replenish the circulating pool about every 12 hours.
(A25) Blood cell losses can be accentuated appreciably under a variety of disease states(including ionising radiation induced cytopenias), and the durability and capacity of themarrow’s blood-cell production are quite impressive. It is estimated that there are ~2600
grams of active bone marrow distributed somewhat unevenly within some 206 bones of the
body (not all bones contain active bone marrow, but under selectphysiological/pathophysiological conditions, all can be induced to produce active marrow).
This marrow contains approximately 1.26 x 1012 bone marrow cells (Fig. A.4.) and has aturnover rate of 3.16 x 1011 cells per 70 kg body weight per day (Fliedner, 1998). From thesenumbers it would appear that in aggregate, approximately a quarter of the marrow renews
itself daily. However, the latter estimate is an average over all the marrow compartments, andturnover within specific compartments (e.g. progenitor versus post-proliferative, maturing
compartments) can vary by orders of magnitude.
(A26) When the haematopoietic system comes under stress and circulating blood cell levels
fall (or rise) abnormally, the marrow responds in a strictly controlled and graded fashion.
First, the tissue releases (or retains, in cases of excess cell numbers) stored reserves of fully
mature functional cells. Second, the tissue alters (shortens in cases of deficit, and lengthens incases of excess) the transit times of maturing, non-dividing cells. Third, there can beadditional divisional cycles within the proliferative cell compartments. Fourth, there can beselective enhancement of differentiation of progenitor cells at the expense of self-renewal.
Experimental data support the contention that all of the latter processes are operative during
or following periods of severe cytopenia, especially those that arise as a consequence of highdose radiation exposure (Carsten, 1984; Fliedner et al., 2002).
(A27) Turnover rates of marrow HSCs have been estimated for a number of species,
including humans. The estimates have been made using indirect approaches, i.e. measures ofdownstream responses of HSC daughter cells as reflections of the true kinetics of HSCs
themselves (Shepherd et al., 2004; Shepherd et al., 2007). Using a combination of techniques,i.e. telomere length measurements using fully mature blood granulocytes and selective
genetic lineage tracing of marrow progenitors, turnover rates of marrow HSCs in different
species have been estimated more accurately. Average HSC turnover rates for the following
species were reported: ~2.5 weeks in mouse (via telomere shortening assays); ~8.3 weeks incat; ~23 to 36 weeks in non-human primate (baboon) (depending on the use of telomere
shortening or gene tracking); and ~45 weeks in humans (Shepherd et al., 2004; Shepherd et82

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al., 2007). From these data, Shepherd et al. suggested that there is a highly conserved,
species-independent constancy for HSC replication over the lifespan of the individual. Thisestimate ranged from 80 to 200 HSC replications per individual per lifespan.
A.3.2. Age dependence(A28) Age has a profound effect on both structure and function (and radiosensitivity) of
the body’s haematopoietic tissues. The earliest, ‘primitive’ form of haematopoiesis is within
the yolk sack (3-4 weeks post-conception), subsequently it moves to the fetal liver (4-8weeks) and only after 8-9 weeks does it start to move to its final destination in fetal bone
(Kelemen and Calvo, 1982). In addition to this initial ephemeral site of haematopoiesis
within the yolk sack, a second, more stable and ‘definitive’ site of haematopoiesis containing
self-renewing, multipotent adult-type HSCs emerges autonomously within theaorta/gonad/mesonephros (AGM) region of the embryo (Tavian et al. 2010). Haematopoiesis
in fetal bone results from the seeding of liver-derived, circulating HSCs into a bonyencapsulated receptive matrix comprised of innervative vascular and mesenchymal elements(Fliedner et al., 2002). The cellular processes and sequence of steps in developing thisreceptive matrix within the primitive bone cavity appear to be similar in most mammals, e.g.
cartilage within fetal bone cavities becomes necrobiotic, leaving centralised cavities into
which primitive mesenchymal elements of the perichondrium penetrate, followed by
infiltrating nerves and vasculature (Fliedner, 1998). The change from skeletal cartilage to prehaematopoietic stroma to haematopoiesis seems to be the same among mammalian species,
although the temporal patterns expressed by different bones appear to differ. The latter
sequence of matrix development to stem cell seeding to haematopoiesis, starts at about 6 to 8
weeks of gestation, but appears not to be completed (all bone marrow sites initiated) until ~15
weeks or later. Only during these later gestational periods (≥16 weeks post-conception) does
the bone marrow become the dominant site of haematopoiesis in the developing human fetus(Kardel et al. 2012). High concentrations of circulating stem cells and progenitor cells, alongwith an abundance of receptive stromal niches within bones, seem to drive the seeding
process and the establishment of new sites of haematopoiesis within fetal bones. By 21-22
weeks post-conception, long bones, ribs and sternum, and vertebra exhibit robust
haematopoiesis (red marrow dominates and yellow marrow is absent). This accounts for~70% of the active marrow and its estimated 1.5-2.5 x 1010 nucleated marrow cells at birth(Kelemen and Calvo, 1982). During prenatal periods (>12 weeks post-conception), themarrow is rich in myeloid-committed progenitors (e.g. GM-CFU), and estimates are in therange of ~2-10 x106. Similarly, fetal blood concentrations of stem cells and progenitor cells
appear enriched, an order of magnitude larger than those concentrations found in adults undernormal steady-state conditions.
(A29) In contrast to the quiescent nature of primitive HSCs from bone marrow of adults,
prenatal progenitor cell populations are non-quiescent and actively cycling; it is only during
the early postnatal period (i.e. ~3-4 weeks in mice and probably ~2-4 years in humans) thatthe “quiescent state” becomes a characteristic feature of marrow HSCs (Bowie et al., 2006).
Further, judging from the report by Bowie et al. (2006), full ‘engrafting’ potential of HSCs isreached only when this active cell-cycling ends, along with the transient overexpression of an
essential, but countervailing HSC-homing chemokine (CXCL12).
(A30) The risk of radiation-induced leukaemia appears to decline with age at the time of
exposure (Table A.4.), but increases with the elapsed time following exposure (BEIR VII,
2006). The relative leukaemic risk within the A-bomb survivor cohort declined appreciably
with increasing age at the time of exposure (Pierce et al., 1996; Preston et al., 2004). In
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contrast, the estimated risk increased with time following exposure. In very young survivors(0-9 years of age), the risk steeply increased during the early years following exposure,
whereas in the older survivors, the rise in risk was significantly delayed and more gradual.
(A31) Estimates of leukaemic risk for the irradiated, developing fetus are less clear thanfor the young or for the adult. Earlier work stemming from a Study of Childhood Cancers in
the United Kingdom reported RRs in the range of 1.4-1.5 for pregnant women who
underwent obstetric x-ray examinations, and had received very low doses estimated to be in
the range of 10-20 mGy (Doll and Wakeford, 1997). Follow-up analyses of these studies havequestioned these elevated ERRs on the grounds of bias and confounding factors (Boice andMiller, 1999; ICRP Publication 90, 2003; NCRP Publication 174, 2014). Similarly,
Delongchamp et al. (1997) reported elevated leukaemia mortality among in utero irradiatedA-bomb survivors. However, this apparent rise in “radiation-associated leukaemia” wasbased on an extremely small, statistically-challenging number of observed cases, so thatchance could not be discounted as an explanation. More recent analyses of leukaemia within
this cohort have failed to detect a significant increase in leukaemia, especially whencompared to the elevated number seen in the postnatally-exposed A-bomb survivors (DLPreston, personal communication; Hsu et al., 2013). Further, in a report of cancer risks (solidcancers and leukaemias) within 8,000 children of female Mayak nuclear workers who were
exposed in utero to low doses (mean doses of ~55 mGy) of ionising radiation, any significant
elevated cancer risks were not shown (Schonfeld et al., 2012). Similar differences in theapparent estimated risk of fetal exposures have also been noted experimentally. For example,
a low incidence or even an absence of induced leukaemia or cancer was found in fetal
irradiated mice (Upton, 1960; Ellender et al., 2006; Di Majo et al., 1990) and irradiatedcanines (Seed et al., 1987), yet increased leukaemia or cancer rates were observed following
postnatal irradiation.
Table A.4 a & b. Age-dependent responses relative to lifetime attributable risks of leukaemia
and other cancers: incidence and mortality1,2
a. Incidence
Cancer type/ gender
Leukaemia/ male
Leukaemia/ female
Solid cancers/ male
Solid cancers/ female

3399	
  
3400	
  

0
237
185
2326
4592

5
149
112
1667
3265

10
120
86
1325
2525

Age at exposure (years)
20
30
40
50
96
84
84
84
71
63
62
62
881
602
564
507
1575
1002
824
678

60
82
57
407
529

70
73
51
270
358

80
48
37
126
177

0
71
53
1028
1717

5
71
52
781
1295

10
71
53
641
1051

20
67
51
444
711

Age at exposure (years)
30
40
50
64
67
71
51
52
54
317 310259
491 455
415

60
73
55
246
354

70
69
52
181
265

80
51
38
102
152

b. Mortality
Cancer type/ gender
Leukaemia/ male
Leukaemia/ female
Solid cancers/ male
Solid cancers/ female

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1
2

Number of cases per 100,000 persons exposed to 0.1 Gy
Data from BEIR VII, phase 2 (2006). Tables 12D-1 & 12D-2 in Annex 12D, pp 311

A.3.3. Cellular features of HSCs
(A32) HSCs and their early progeny, i.e., haematopoietic progenitor cells (HPCs), aregenerally characterised either by (a) their functional attributes, (b) their cell surface properties,
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or (c) a combination of these. This is in contrast to the conventional morphologicaldefinitions commonly applied to the fully mature and functional blood cells within thecirculation. The most primitive of the HSCs (independent of species of origin) is not clearly
identifiable morphologically. They have rather primitive, monocytic-like features, notdissimilar to that of small lymphocytes. These HSCs are small, roundish cells, ~7-10 µm in
diameter, with large, oval to round nuclei, small cytoplasmic rims, and generally relatively
smooth cell surfaces (van Bekkum, 1976). HPCs tend to be a little larger and somewhat more
pleomorphic in size and shape, often reflecting cellular features characteristic of lineage
commitment.
(A33) Functional descriptors: HSCs can be routinely identified, enumerated, andcharacterised using functional assays that utilise basic characteristics that serve to define the
HSC’s self-renewing and cell-amplifying properties and its potential for differentiation.
Invariably, these assays are indirect by nature, employing the serial dilution and transfer oftest marrow cells or marrow cell fractions into recipient animals or cultures, with subsequenttime-based monitoring of either (a) tissue repopulation, or (b) tissue or animal survival as
endpoints.
(A34) Lineage-committed HPCs, by contrast, can be quantified and functional properties
examined somewhat more directly through the use of colony assays performed either in vitro(CFU-C) or in vivo (CFU-S) (Metcalf, 1971, 1977). Serial dilution and transfer plating (orinfusion) of HPC-enriched cell fractions, with subsequent monitoring of clonogenic
responses are exceedingly useful procedures for determining the numbers, proliferative anddifferentiative potentials of marrow-derived HPCs, regardless of their multi-, bi-, or unilineage potentials. These assays have provided, and continue to provide, an opportunity to
examine and assess in detail the function of both individual and populations of HPCs.
(A35) Assays of myeloid-committed HPCs have long been used as clinically relevant,“surrogate” markers of marrow HSCs within both donor and recipient marrow prior to, or
following, autologous or allogeneic bone marrow transplantation (Mangalik et al., 1979).
(A36) Cell surface characteristics: With the advent of multicolour FACS protocols, andarrays of cell-type specific monoclonal antibodies, phenotying via cell surface markers ofrare lympho-haematopoietic progenitors was made possible (Spangrude et al., 1988).
Currently, HSCs and HPCs can readily be distinguished using flow cytometry. Specificsubsets of HSCs and lineage-specific HPCs can be identified, counted, and analysed in both
steady-state conditions and after exposure to physiochemical toxicants (e.g. benzene andionising radiation), as well as in evolving pathological conditions.
(A37) The major distinguishing cell surface features of these main classes of lymphohaematopoietic progenitor cells are as follows. First, progenitor cells of the marrow, i.e.
HSCs plus HPCs, are characterised by a select few surface markers. For example in mice,high levels of Sca-1+ and stem cell factor receptor (cKit+) along with low levels of thymocyte
surface antigen-1 (Thy-1lo) and low levels of CD34+/lo, define the progenitorial class, whereas
in primates, high levels of the CD34+/hi marker serves to define the progenitor cell group(NIH Report, 2008). Other defining surface markers for select HSC subsets in primates havebeen reported as well. These markers include, but certainly are not limited to, KDR+ which isa VEGRF2 surface receptor (Ziegler et al., 1999), the SLAM family receptors (Kiel et al.,2005), and myeloid leukaemia 1 (MCL-1) which is an apoptosis response modulator thatbelongs to the BCL-2 gene family (Opferman et al., 2005). Further, an HSC surface adhesion
molecule, CD49f, has been recently described as a specific HSC marker in humans; HSCs
bearing CD49f were characterised as being highly efficient in generating long-term,
multilineage grafts (using an optimised HSC xenograft assay of intrafemorally injected,
highly-enriched HSCs into female NOD-scid-IL2Rgc-/- mice) while the loss of the surface
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marker identified a transiently-engrafting multipotential progenitor (MPP)-cell subpopulation(Notta et al., 2011). Second, HSCs are defined as being “lineage negative (Lin-), whereas
HPCs are defined by lineage-positivity (Lin+) for either all, or some of specific lineages
identified using a battery of monoclonal antibodies directed to specific blood cell types. Othersorting strategies using different arrays of cell surface markers have also been successfully
employed. One sorting strategy used the differential expression of the surface marker “Rho”
on HPCs and HSCs (Eckfeldt et al., 2005). Low Rho (Rholo) expression on CD34+CD33CD38- cells served to define the HSC population, whereas the high Rho expression (Rhohi) on
CD34+CD33-CD38- cells identified them as HPCs. In turn, these sorted HSCs and HPCs wereused in a unique functional screen (a morpholino antisense oligonucleotide-based screen inzebrafish) to identify and assess essential function of HSC- and HPC-specific genes during
very early stages of organogenesis.
(A38) Nuclear proteins and genes: As HSCs reproduce and also produce progenitor
daughter cells committed to either myeloid or lymphoid lineage development (HPCs), silent
or low-functioning lineage-specific genes become activated (Krause, 2002). HSC-to-HPC
transition and the associated shift from a non-lineage-committed state to one that iscommitted, occur when lineage-associated genes are activated by selective demethylation of
specific lineage-repressive amino acids within bounding histones. At the macromolecularlevel, regions of condensed chromatin become more open and permissive of active
transcription of associated genes. Good examples of the latter are data indicating that: (a) the
activation of normally-silent B- and T-lymphoid related genes within human HSCs
(CD34+CD39lo) is through the selective loss of repressive methylation marks on lysine 9; and(b) selective demethylation of H3 lysine 4 within B-cell specific gene loci (Maes et al., 2008).
(A39) There is general support for the concept that lymphoid-specific genes are already“primed” for expression prior to lineage commitment. A continuum of molecular events or
stages exists within HSCs during the priming and full commitment to transition to HPCs. In
contrast, HSC/HPC transition-related commitment either to erythroid, granulocyte and T-celllineages appears more global and less specific, with loss of histone acetylation at nonlineage-associated genes. Nevertheless, the essential nature of activating select types orclasses of genes in lineage commitment and subsequent lineage development has been well
documented: e.g. c-Myb for T-cell development (Allen et al., 1999), and calmodulindependent protein kinase (CaM kinase) IV in regulating erythroid lineage commitment andsurvival (Wayman et al., 2000).
(A40) Cytokines, cytokine receptors and cell signalling: The precise nature of HSC selfrenewal and lineage commitment remains ill defined. Regulation of HSC involves a
combination of extrinsic and intrinsic signalling pathways that need to converge in order toproperly regulate HSC’s decisions on whether to self-renew or to initiate differentiation (Fig.
A.2.) (Eckfeldt et al., 2005). Although a large number of cytokines, chemokines, andrespective surface receptors have been identified, cloned and analysed for targeting activity
and specificity, none has been demonstrated to have exclusivity relative to these vital “fatedecision responses” by the HSC in a steady state as well as following irradiation. Nontheless,a number of novel regulators of HSC fate decisions have been identified. Overexpression of
homeobox b4 (Hoxb4) has been shown to enhance replication of both murine and humanHSCs, and in turn, to increase their repopulation potential (Antonchuk et al., 2001; Buske etal., 2002; Sauvageau et al., 1995). In contrast, maintenance of HSC quiescence appears to be
intrinsically controlled, at least in part, by p21, a G1-checkpoint, cyclin-dependent kinase
inhibitor (Cheng et al., 2000). Extrinsic regulators of HSC self-renewal have been identified,including Notch (Varnum-Finney et al., 2000), mammalian homologues of Drosophila

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wingless (Wnt) (Murdoch et al., 2003; Reya et al., 2003), sonic hedgehog (SHH) and bonemorphogenetic protein 4 (BMP4) (Bhardwaj et al., 2001).
(A41) In situ, HSCs reside within growth-regulating stromal-cell niches of the marrow.
“Fate decisions” made by resident HSCs represent in part the net result of signalling
pathways emanating from the niche. A host of haematopoietic stimulators, e.g. cytokines,
growth factors, soluble cytokine/growth factor receptors, such as SCF, fms-related tyrosine
kinase 3 (flt3), erythropoietin, thrombopoietin, interleukin (IL) 6, soluble IL6 receptors, havebeen shown to enhance HSC and early HPC proliferation (Henschler et al., 1994; Zandstra etal., 1997).

3515	
  

A.4. Radiosensitivity

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(A42) HSCs and lineage-committed progenitor cells (HPCs), as well as moredifferentiated, proliferative daughter cells, all exhibit a high degree of heterogeneity
regarding radiosensitivity (Wagemaker, 1995). The origin of this heterogeneity is not entirely
clear, owing to the considerable differences in composition and function of largely-undefined
subsets contained within these larger, progenitor haematopoietic compartments.
Radiosensitivities are dependent on the species (e.g. rodents, canines, primates), the subclassof cells being tested, cell-cycle status, growth factor and microenvironmental influences(Hendry and Yang, 1995). In mice, these relationships have been well studied anddocumented, and hence are fairly well accepted as being accurate and reflective of thesituation in other mammals, including primates. As one moves from the most primitive of the
HSCs in the marrow (cells with marrow repopulating ability, MRA cells) to slightly lessprimitive, multipotential, lineage-committed cells such as CFU-S-day-12, to more mature,lineage-restricted HPCs (G-CFU or GM-CFU), the radiosensitivity of these cells shifts, anddramatically so, but not in a unidirectional manner (Gidali, 2002; Imai and Nakao, 1987;
Ploemacher et al., 1992; Scheding et al., 1996). Further, minor subpopulations ofradioresistant cells within larger, more dominant radiosensitive HSC and HPC marrowcompartments have been clearly identified and documented within marrow of a variety of
species, including mice, dogs, and primates (Inoue et al., 1995; Seed et al., 1982; van
Bekkum, 1991). Based on a continuous monitoring of such shifts in radiosensitivity of
myeloid-committed progenitor cells in marrow of chronically irradiated dogs, it has beensuggested that these small radioresistant subpopulations become dominant following
extended periods of radiation exposure, often associated with evolving myelodysplasia and/or
frank myeloid leukaemia (Seed et al., 1985).
(A43) Shifts in radiosensitivity during very early phases of lineage committment (e.g.
from quiescent, long-term marrow repopulating HSCs to short-term, non-quiescent,repopulating and cycling multipotential/multilineage progenitors) are associated with equally
dramatic shifts in terms of type of basic DNA repair pathways employed to repair potentiallylethal DSBs following acute radiation exposure. Specifically, the most primitive and mostquiescent of the marrow HSCs (in adult mice) appear to employ mainly error-prone NHEJ,
whereas MPPs employ a high fidelity HR during cell reproduction in order to correct major
lesions (Mohrin et al., 2010).
(A44) Radiosensitivity tends to rise during early HSC-to-HPC transition, and associatedearly phases of lineage commitment, and sequentially tends to fall selectively with further
lineage-restriction and progenitor cell maturation (Fig. A.5.). Meijne et al. (1991) showedthat within mouse marrow progenitor cell populations, (1) MRA cells are moderately
radioresistant with measured D0 values in the range of 1.13-1.18 Gy; (2) less mature CFU-87

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Sday12 and more mature CFU-Sday7, both with multilineage potentials, have D0 values of 0.94
Gy and 0.71 Gy respectively; and (3) more mature lineage-restricted bi- or uni-potential
myeloid progenitor cells have significantly higher D0 values (~1.2-1.6 Gy). In contrast,
murine erythroid lineage-restricted progenitors are considerably more radiosensitive withlower D0 values: 0.68 Gy for burst-forming units erythroid (BFUe) and 0.53 Gy for colonyforming units erythroid (CFUe) (Imai and Nakao, 1987).
(A45) Shapes of ‘radiation dose-cell survival’ curves are fairly consistent for the above
mentioned progenitor subtypes within various mammalian species irradiated under steadystate haematopoiesis. In general, the decrease in survival of either HSCs or HPCs tends to be
exponential with increasing radiation dose, without a substantial threshold at the lowest doses.
This common pattern is illustrated in Fig. A.6a. by the single-dose/response relationship for
CFU-Sday9 (Bond, 1995). Further, relationships between the extent of radiation-induced lossof these vital progenitor cell types, the overall severity of marrow injury, and 30-day
mortality of the irradiated animals are illustrated in the complementary Fig. A.6b. Thesefigures highlight the fact that only a very small fraction of surviving progenitors is required tomaintain organ function. For survival of the larger species (man, dog, Rhesus monkey), a
significantly greater surviving fraction of these vital marrow HSCs/HPCs after irradiation isrequired, than for the smaller rodent species (mouse, rat) (Vriesendorp and van Bekkum,1984).
(A46) Despite the very common features of the dose/response patterns of the various
progenitor cell subtypes, specific differences do exist and largely relate to dose-dependent
rates of cell lethality (as reflected by differences in the slopes of the survival responses). Fig.
A.7a-d shows representative “radiation dose-survival response” patterns, along withestimated “D0 values” of several major progenitor subtypes from B6D2F1 male mice 24 hours
following acute, whole-body, γ ray (137Cs) exposures.
(A47) Haematopoietic disequilibria, regardless of aetiology, will result in marked shifts inassayable, progenitor cell survival as well. For example, growth factor-mediatedmyelopoietic stimulation of HSCs/HPCs will elicit marked changes in both sublethal andpotentially lethal damage capacities (Fitzgerald et al., 1989; Goff et al., 1997). Similarly, thedisequilibria associated with chronic, low daily dose γ irradiation (7.5 cGy day-1) ofexperimental canines fosters outgrowth and repopulation of normally minor, radioresistant
subsets of myeloid progenitors (Seed et al., 1982).
(A48) Similar shifts in radiosensitivity of human HSCs/HPCs have been noted as well.
However, the results are not quite as clear due to a variety of technical limitations in testing(e.g. relatively low purity of test cells, coupled with low plating efficiencies in vitro).
Nevertheless, workers such as Kreja et al. (1993) reported on the in vitro growth
characteristics and the radiosensitivity of CD34+ enriched cells from human umbilical cord
blood. Following immunomagnetic bead selection, CD34+ cells were irradiated over a fullrange of doses with x-rays, plated under clonogenically-permissive growth conditions(complete tissue culture media, supplemented with a cytokine cocktail containing
recombinant human SCF or bFGF, erythropoietin, and placenta conditioned medium, andassessed for the types and numbers of specific colonies formed. Not surprisingly andconsistent with work done in rodents, the less mature, multipotential HPCs were considerably
more radiosensitive than the more mature, lineage-restricted HPCs (e.g. mixed colonyforming cells had D0 values ~95 cGy, whereas the uni- or bipotential BFUe and GM-CFU
had D0 values of ~136 cGy).

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Radiosensitivity:	
  HSCs	  and	
  HPCs
1.6
1.4
1.2
1

Do	
  values

0.8
0.6
0.4

0.2
0

Hematopoietic	
  progenitor	
  subtypes
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Fig. A.5. Radiation sensitivities (measured in terms of D0 values) of various murine HSC and HPC
subsets are shown. HSCs and HPCs, as well as more differentiated proliferative daughter cells all
exhibit a high degree of heterogeneity regarding radiosensitivity. D0 values are variable: primitive
repopulating HSCs (MRA and longterm cultured cells (LTC-Ic)) are relatively radioresistant;multipotential, lineage-restricted spleen colony-forming cells (CFU-S assayed at day 12 or 9) are lessresistant; whereas multi- or bipotential, granulocyte/erythroid/monocyte/megakaryocyte lineagerestricted in vitro colony-forming cells (GEMM-CFU and CFU-meg) are more resistant compared toerythroid-restricted progenitors (BFUe or CFUe). Data were taken from the following references(Gidali, 2002; Imai and Nakao, 1987; Ploemacher et al., 1992; Scheding et al., 1996; Testa and Lajtha,
1973; Till and McCulloch, 1961). (permission needed)

Fig. A.6. Left panel: A representative radiation dose/ haematopoietic progenitor cell survival responseis shown (with survival plotted on a log-linear scale). Right panel: An inverse relationship of thedeclining survival of marrow CFU-S and rising severity of haematopoietic tissue damage is shown
relative to a lower range of radiation doses, whereas a rapidly rising incidence of mortality is shown89

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to occur at a significantly higher range of radiation doses (Bond, 1995). (Figures reproduced withcourtesy and permission from John Wiley & Sons Inc.) (permission needed)(A49) When the radiosensitivities of three major HSC/HPC classes, namely MRA, CFUSday7, and CFU-C, were assayed both in vivo and in vivo and under different levels ofoxygenation, following either 1 MeV fission neutrons or 300 kVp x-rays, the differences inradiosensitivity expressed by these maturationally-distinct progenitor cells were found to be
due largely to intrinsic factors and not extrinsic factors such as niche sites of progenitor
localisation or oxygenation (Meijne et al., 1996). However, later work showed that the mostprimitive stem cells were protected from low-LET radiation by the hypoxic nature of theniche (Kubota, 2008; Parmar et al., 2007).
(A50) The multipotential cells (CFU-S) show little dose-rate effect, and this is consistent
with their fairly high sensitivity to low acute doses which can be characterised by a large αkill component in the LQ formalism. This lack of dose-rate effect was observed even down to
2 decades of depopulation caused by irradiation at 0.45-0.9 Gy/day continuous irradiation(Chu-Tse and Lajtha, 1975), but after an accumulated dose of 2.5 Gy, there was a plateau in
numbers followed by a dose-rate-dependent repopulation rate. Similarly, effects of dose
fractionation are small, until concomitant protraction of exposure results in marked
repopulation effects (Hendry and Lajtha, 1975). Animal studies have shown that thehaematopoietic system is capable of maintaining an adequate number of cells during chronic
low-dose and low-dose rate radiation exposure. This is due to increased rates of cell
production resulting from shortening of the cell cycle and maturation time, increased
proliferative activity of stem cells and precursor cells, and overall stimulation of
haemopoiesis (Fliedner et al., 2002; Gidali, 2002; Lord, 1965).
(A51) The haematopoietic microenvironment, which normally maintains homeostasis of
the stem cell pool by interaction with stem cells and multipotent progenitor cells, plays animportant role in recovery after damage (Molineux et al., 1987). Extramedullaryhaematopoiesis and migration of HSCs from bone marrow to the spleen, liver and lymph
nodes can also occur. Recovery of haematopoiesis is more complete after exposure at low
dose rate than at high dose rate. For example in mice, recovery of haematopoietic and stromalprogenitor cells was almost complete by one year after 12.5 Gy delivered at 0.0005
Gy/minute compared with incomplete recovery after only 6.5 Gy given at 0.7 Gy/minute(Gallini, 1988). Nonetheless, in other studies after low-dose rate exposure, CFU-S was notrestored to baseline levels during the lifetime of the animals, demonstrating some very longterm residual injury (as reported in ICRP, 2012).

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a

b

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Fig. A.7. Representative radiation dose-survival response patterns for various murine haematopoietic
progenitor subtypes: (a, upper left panel) colony-forming units assayed in spleen at day 9 (CFU-sday9);(b, upper right panel) erythroid colony-forming units assayed at day 3 (CFUe3); (c, lower left panel)megakaryocyte colony-forming units (CFU-m); (d, lower right panel) granulocyte/macrophagecolony-forming units (CFU-gm) (Nakeff, 1979). Note the common features of dose-response linearity,
lack of a substantial response threshold, and the differences in the slopes of the dose-survival
responses. (Figure reproduced with courtesy of Springer Inc.) (permission needed)

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A.5. Characteristics of single-cell responses

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(A52) Strategies that employ FACS isolation of potential HSC/HPC marrow cell targets
based on cell surface determinants, followed by in vitro plating of single cell targets bearing
defined surface markers, and subsequent time-dependent, single cell growth, clonal,
biochemical and/or molecular analyses have been reported. CD34+ Lin- bone marrow cells
were FACS isolated and analysed for molecular signatures of myeloid and erythroid celllineages. Approximately 50% of CD34+ Lin- marrow cells expressed mRNA for both βglobin and myeloperoxidase, suggesting that a large fraction of these marrow HSCs were
simultaneously primed at the gene level for both erythroid and myeloid lineage commitment

c

d

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(Hu et al., 1997). It has been suggested that such low-level priming of lineage-specific genes
across multiple lineages is a characteristic of HSCs, (Krause, 2002). Also that full lineage
commitment is more a function of suppressing low-level gene activity within those lineagespecific gene sets not selected for, rather than specifically activating and enhancing geneactivities within the lineage that is ultimately selected for commitment. The influence ofradiation exposure on these pre-commitment processes is unknown.
(A53) A fundamental regulatory control of HSCs’ cycling status and lineage-commitment
process(es) is exerted directly by local environmental signals generated by the bounding
stromal niche (Trentin, 1989). Further, the niche appears to contribute not only to the HSC’s
repopulating capacity, but also to the maintenance of quiescence, thus allowing these cells tostay in a radioprotective, noncycling state (Greenberger and Epperly, 2009). The more
importance of these HSC-regulatory signals certainly includes the Tie2/Ang-1 signalling
pathway. In this pathway, the stromal cell niche supplies the functional ligand, Ang-1, thatinteracts with specific tyrosine kinase surface receptors, Tie2, on HSCs in order to maintainnot only the stem cell-defining features of quiescence and self-renewal, but also adhesivenessto key stromal elements (e.g. osteoblasts) within the niche itself (Arai et al., 2004). Other
niche-associated factors, e.g. oxygenation, also play key roles in preservation of “stemness”(Parmar et al., 2007; Suda T, 2011).

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A.6. Mutagenesis

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(A54) Mutagenesis of the blood-forming system of mammals, including man, is wellfounded and documented (Bartel et al., 2000; Caddle et al., 2008; Germeshausen et al., 2008;
Mattison et al., 2007). Although most mutations can be found within fully mature, circulating
blood cells, these mutated cells originate largely within the progenitor cell compartments ofthe marrow. Mutations within HSCs and HPCs appear to arise either spontaneously, or
following exposure to a wide variety of physiochemical toxicants and/or biological agents.
Also, all progenitor cell classes have the potential to serve as targets for ionising radiationassociated mutagenesis. Mutant subpopulations of fully mature, non-proliferating blood cells(e.g. glycophorin A (GPA)-mutated erythrocytes, PIG-A-mutated leukocytes) have been
detected within the circulation of individuals long after A-bomb exposure (Dong et al., 2001;
Kusunoki et al., 1995; Kyoizumi et al., 1996; Kyoizumi et al., 2005). This implies (a) alineage-specific nature and progenitor origin of these mutations; and (b) lineage-committedprogenitors are the targets for the mutational process. The doubling dose for GPA mutations
has been estimated to be ~1.2 Sv, with a threshold dose of ~0.24 Sv (Kyoizumi et al., 1996).
A sizeable threshold dose for these GPA-related mutations is most likely responsible forlargely negative findings of a number of investigators who have used the GPA assay tomonitor mutations within erythrocytes of individuals with a prior history of very low
radiation exposures (Tawn et al., 2003).
(A55) The timing and expression of radiation induced mutations within haematopoietic
progenitor cells have been investigated (Kadhim et al., 1994; Kadhim et al., 1992; Kadhim
and Wright, 1998; MacDonald et al., 2001; McIlrath et al., 2003; Wang et al., 1996; Watson
et al., 1996; Watson et al., 2001). A delayed expression was reported of nonclonal
chromosomal aberrations within colonies of non-irradiated haematopoietic cells that had been
derived from irradiated haematopoietic progenitor cells many cell generations earlier(Kadhim et al., 1994; Kadhim et al., 1992). Initially, these delayed mutations were thought tobe produced only by high LET α particle irradiation, but later were shown to occur with otherradiation qualities (x and γ rays) (Watson et al., 2001).

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(A56) These delayed genomic lesions are now considered part of a larger class ofradiation-associated genomic responses, commonly referred to as “non-targeted” effects.
These include both manifestations of ‘genomic instability’ and ‘bystander responses’ within
downstream non-irradiated haematopoietic daughters of the original irradiated parental cell orof haematopoietic progenitor cells residing in close proximity to irradiated neighbouring cells(Wright, 2010). Wright and colleagues have shown that at least within thelymphohaematopoietic system, these delayed responses are highly ‘genotype-dependent’ andtied to aberrant molecular signalling related to inflammatory processes. Specifically, thesesignals (i.e. partially identified as tumour necrosis factor α, nitric oxide, and superoxide) havebeen shown to emanate from heavily, but not lightly irradiated (bystander) macrophages(Lorimore et al., 2008; Zyuzikov et al., 2011).
(A57) The role of radiation-induced genomic instability in humans is uncertain. Kodama
et al. (2005) evaluated in detail the cytogenetics of blood T-cells of A-bomb long-term
survivors and found no evidence to support the concept of exposure-induced chromosomal
instabilities. Nakamura (2005) speculated that the radiation-induced leukaemias within the Abomb surviving cohort might be associated with a very small subset of individuals,
predisposed to the radiation effects by virtue of pre-existing, naturally-acquired chromosomal
lesion(s). Delayed-type Hprt mutation frequencies were consistently and significantly
elevated by prior irradiation of stem cells (more likely a mix of multipotential and more
lineage-restricted HPCs) (Harper et al., 1997). Elevated mutation frequencies were noted not
only for α particle irradiation, but also for 250 kVp x-rays, and even more markedly for fastneutrons. Further, mutation frequencies of the delayed type of mutations were consistently
elevated, albeit marginally for the neutron and x-ray induced mutations, relative to the more
immediate-type of mutations. The estimated mutation frequencies per Gy for both theseclasses of mutations are listed in Table A.5. (Harper et al., 1997).
(A58) Radiation mutagenesis within more primitive haematopoietic progenitors (e.g.
marrow repopulating stem cells) is less well defined. Long-persisting chromosomal
instability within fully reconstituted marrow of recipient mice was detected following an
earlier (1 year) total-body irradiation conditioning and subsequent syngeneic transplantationof in vitro, α-particle-irradiated marrow repopulating stem cells (Watson et al., 1996). Longterm radiation-induced chromosomal instability was also reported within Lt-HSCs ofrepopulated marrow of adult Swiss mice given 0.5-1.5 Gy of γ rays while in utero (Devi andSatyamitra, 2005). The dose range used in this study and the long-term mutational effects
noted provide a rough estimate of the radiosensitivity (relative to mutagenesis) of marrow
repopulating Lt-HSCs in mice.

1

Table A.5. Estimated Hprt mutation frequencies for irradiated marrow HPCs 1,2
Radiation type/ quality
Hprt mutation frequency per Gy
Immediate
Delayed-6
α particles
9.6 x 1018.0 x 10-6
Neutrons
21.3 x 10-6
26.3 x 10-6
-6
x-rays4.6 x 10
6.9 x 10-6

Estimates based on raw mutation frequencies (Harper et al., 1997) and with assumed linearity
relative to radiation dose delivered.
2
Background mutation frequencies of unirradiated mouse marrow HPCs were 2.6 x 10-6 and 4.5 x
10-6 for immediate- and delayed-type Hprt mutations, respectively.

(A59) In contrast to the little information concerning radiation mutagenesis of primitive
marrow HSCs, information on radiation mutagenesis within the lymphocytic cell lineage,

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especially circulating blood lymphocytes, is relatively plentiful, and clearly instructive interms of the nature of radiation mutagenesis within the lympho-haematopoietic system. For
the induction of Hprt mutations in splenic T lymphocytes of 137Cs γ irradiated mice, themutation frequency depended markedly on dose, dose rate and time after exposure (Lorenz etal., 1994). Dose-response relationships for Hprt mutations were best fitted at high dose rateby an LQ function (i.e., MFs (high dose rates) = 6.9 x 10-6 Gy + 1.2 x 10-6 Gy2), whereas at low
dose rates, a simple linear function seemed best suited (i.e. MFs(low dose rates) = 3 x 10-6). The
DDREF was 1.5 for doses of <2 Gy and dose rates of 1 Gy/day or less, and 3-5 for high doses
and dose rates (Lorenz et al., 1994).
(A60) Kataoka et al. (1993) reported the frequencies of Hprt mutations induced by both
single and fractionated doses of fission-spectrum neutrons and compared those estimates with60
Co γ ray induced mutations. The estimated mutation frequencies for single, acutely
delivered doses (1.5 Gy) of fission neutrons were not significantly different from theestimated mutation frequencies for single doses (7.5 Gy) of 60Co γ rays (i.e. (5.98 ± 1.51 SE)
x 10-5 versus (5.56 ± 3.09 SE) x 10-5). By contrast, the fractionated regimen of fission
neutrons (0.25 Gy x 6, to total doses of 1.5 Gy) elicited marginally, but not significantly
higher mutant frequencies compared to comparably fractionated doses (1.5 Gy x 6) of γ raysdelivered to total doses of 9.0 Gy: estimated mutation frequencies for fractionated neutron
exposures = (8.71 ± 5.39 SE) x 10-5; MFs for γ-rays = (2.30 x 10-5) ± (9.07 x 10-6) SE. Not
surprisingly, these results support the concept that the genotoxic potency of high LET fission
neutrons, relative to low-LET γ-rays, appears to increase, albeit marginally, with fractionatedexposure regimens.
(A61) Consistent with the above mentioned reports, Griffiths et al. (1994) compared theestimated Hprt mutation frequency of x irradiated B-cell precursors with otherhaematopoietic cell types and found them to be quite comparable. Interestingly, in a latterstudy (Griffiths et al., 1997), these workers found that the apparent high levels of Hprt
mutations within x irradiated p53 null cells were the result of a preferential survival of themutant, p53 null cells, rather than from a p53-dependent increase in mutation rate. Thisobservation clearly highlights the need for due caution when interpreting these estimatedmutation frequencies.
(A62) Over a decade ago, Kadhim and Wright (1998) noted that “...delayed chromosomal
abnormalities, delayed cell death by apoptosis and late-arising specific gene mutations may
reflect diverse consequences of radiation-induced genomic instability”. They also pointed
that the relationship(s) between these radiation-associated changes was not well established,
and that “…the expression of these delayed heritable effects is determined by the type ofradiation exposure, type of cell and a variety of genetic factors…”. In terms of radiationinduced leukaemia, it is clear that this type of non-specific genetic damage abounds withinradiation-induced leukaemic cells, and is attributable to the accumulation over time of genetic
lesions within irradiated HSC (and/or early HPC) targets. This is, of course, compatible withthe general concept that radiation leukaemogenesis is a multistage process, requiringextended processing/development times, involving genetic instability, with subsequent stem
cell crisis and premalignant/malignant transformation(s) (MacDonald et al., 2001; Seed et al.,
1989; Seed, 1991).
(A63) How a specifically identified genetic lesion, or a collection of genetic lesions,
within marrow cell targets at defined post-exposure/preclinical periods, relates to thepenultimate leukaemic cell transformation, remains ill defined. Some light has been shed into
this “black box”, however, by virtue of the progress made in understanding the nature andaetiology of CML, and the role played by a key gene alteration, namely the bcr/abl gene and94

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its p210 product. This gene is formed by an aberrant reciprocal translocation of chromosomes
9 and 22 to form the pathonomic “Philadelphia chromosome” and its unique fusion gene(bcr/abl) and gene product (p210). By limiting the formation or controlling function of thisgene, one can control the disease, regardless of its aetiology (spontaneous, radiation-, orchemical-induced). The chimeric gene, bcr/abl, can be directly induced within targetedhaematopoietic cells in vitro by very high (i.e., 50-100 Gy) doses of low-LET irradiation (Itoet al., 1993b; Spencer and Granter, 1999), thus providing direct evidence of its induction by x
irradiation in vivo. However, the relevance of the latter studies has been seriously questioned,
and rightfully so, as the “inductive” doses used in these experiments are at least an order ofmagnitude greater than what one would normally perceive as being “biologically relevant”.
Clearly, these observations, however interesting, need to be confirmed at a much lower rangeof radiation doses.
(A64) It is interesting to note that Ito and his colleagues (1993) reported that these
artificially-created fusion genes contained not only CML-specific bcr/abl rearrangements, butalso other forms of bcr/abl fusions as well: e.g. fusion genes with junctions of bcr exon 4/abl
exon 2 intervened by a segment of DNA of unknown origin, bcr exon 5/abl exon 2, and bcr
exon 4/abl exon 2. These authors speculated that only those progenitor cells that bear certain
CML-related bcr/abl fusion genes are positively selected by virtue of a growth advantage invivo. Further, the results of the Spencer and Granter study (1999) showing significantly
elevated levels of bcr/abl transcripts within in vitro irradiated leukaemic cells, whencompared to the lower frequencies found in comparably-irradiated non-leukaemic cells,
prompted the suggestion that the radiation-induced illegitimate recombination may be due toaberrant DSB repair mechanisms within individuals predisposed to CML.
(A65) Other leukaemia-type specific chimeric genes (e.g. the AML t(8,21)-associatedchimeric gene, aml1/eto) have been shown to be directly radiation-inducible as well(Deininger et al., 1998). Further, these hybrid mutant genes appear to originate within
multipotential, trilineal-committed marrow progenitors (Miyamoto et al., 1996).
(A66) Despite the intriguing and insightful nature of the bcr/abl story, the consensus isthat leukaemic cell transformation by ionising radiation, or any other known leukaemogen, is
not merely a function of an aberrant, single gene- chimeric or otherwise; but rather that thetransforming process is both multigenic and multi-staged by nature (Fig. A.8.). Although it isstill relevant to think of radiation leukaemogenesis in terms of cooperative networks ofaberrant proto-oncogenes (e.g. Ras, Myc, Myb) and tumour suppressor genes (p53, Rb, etc),
the role of chimeric genes, global genomic instabilities, bystander effects, altered or loss ofexogenous, stromal niche-mediated growth controls, etc. need to be considered as well.

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Fig. A.8. Potential cell targets, processes and lesions associated with radiation leukaemogenesis(NCRP-150, 2005). (Figure reproduced with courtesy and permission from NCRP.) (permission
needed)(A67) Although it is beyond the scope of this report to detail various genes and theirinterplay during leukaemia development, a select few examples are given below in order toillustrate the latter points. First, the cooperative action of c-Myb and bcr/abl fusion gene on
the clonal capacity of two subclasses of marrow progenitors, i.e. Lin- sca-1+ and Lin- sca-1+
Kit+ progenitor cells, has been recently documented (Lidonnici et al., 2008). With allelic lossof c-Myb, normal colony-forming capacity was markedly suppressed in progenitor cells
expressing p210(bcr/abl), while the clonal response was only modestly affected in the nonp210(bcr/abl)-expressing progenitor cells. The results support the contention that c-Myb isrequired for p210(BCR/ABL)-dependent leukaemogenesis. Second, CD34+ progenitor cells
bearing the bcr/abl chimeric gene exhibit a wide array of gene-based functional alterations
linked either directly or indirectly to the CML progenitor phenotype (Kronenwett et al., 2005),
e.g. increased cell-cycling and proteasome activity, downregulation of DNA repair proteinsand detoxification enzymes, decreased expression of CXC chemokine receptors, and up
regulation of GATA-2, etc. The upregulation of GATA-2 is particularly interesting, as it
might provide the basis of enhanced self-renewal of targeted pre-leukaemic HSCs. Third, a
recent study of gene expression analyses of AML blood cells by Casas et al. (2003) provides
additional support for the above mentioned concept of a cooperative imbalance between aselect set of activated proto-oncogenes (e.g. Jun, growh factor receptor-bound ptotein 10:
GRB10) that are upregulated and enhance cell cycling and cell proliferation, and another setof tumour suppressor genes (e.g. p53, p16). The latter normally act in a countervailingmanner, but during patent AML, they appear to be downregulated, promoting genomic
instability and deregulated apoptotic pathways. Comparable changes are readily envisioned
during radiation-induced AML as well.
(A68) A number of candidate gene targets have been identified as well, and these appear
to be associated with radiation leukaemogenesis (in mice), e.g. the HLX1 homeobox genewhich is essential for haematopoietic development, and RAD51, a DNA repair gene, etc. A

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combined analysis was performed on the latter genes, i.e. RAD51 and HLX1 variant alleles,
and a synergistic 9.5-fold increase (95% CI: 2.22, 40.64) in the risk of radiation-induced
AML was observed (Jawad et al., 2006).
(A69) Radiation-induced genetic instability has been documented both at the molecularlevel and at the chromosomal level. For example, Plumb et al. (1997) reported significantly
elevated instability of the Y-chromosome within haematopoietic cells of male mice withacute, radiation-induced leukaemia. Similarly, elevated frequencies of chromosome-2
aberrations, along with associated interstitial deletions, are common cytogenetic findings inacutely-irradiated, leukaemia-prone CBA mice (Bouffler et al., 1996). However, theformation of these aberrations does not seem to determine the individual mouse’s
leukaemogenic sensitivity, since only 20-25% of these irradiated animals eventuallyprogressed to patent AML. This would imply that these radiation-induced chromosome-2
lesions are indeed associated with early pre-leukaemic events (perhaps acting as “initiating
events”), but clearly they are not self-sufficient for leukaemogenesis (in the 3 Gy irradiated
CBA/H male mouse). Further, the radiation-induced chromosome-2 lesion appears not to berate limiting in terms of leukaemogenesis. In this regard, the work by Rigat et al. (2001) is
instructive: using a PCR-based molecular approach, these workers showed that the frequencyof the radiation-induced “loss of heterozygosity” (LOH) of chromosome 2 within stem cellsof CBA/H x C57BL/6 F1 mice was no greater than the induced LOH in other control
chromosomal regions evaluated.
(A70) So what are the rate limiting steps? It has been suggested that the absolute numberof marrow stem cells defines the number of potentially radiation transformable genomes, andin turn, overall leukaemogenic risk (Jawad et al., 2007). If this is the case, then it is thenumber of available targets within the defined radiation volume that defines the leukaemic
rate, and in turn the efficiency of the irradiation. Further, if the available “targets” include not
only the very rare, primitive, endosteal layer-associated stem cells (Lt-HSCs), but also themore plentiful, centrally-located progenitor/daughter cells (e.g., St-HSCs, early HPCs), thisshould substantially favour any/all of the radiation-associated stochastic events of the earlyleukaemogenic process.
(A71) In the light of this possibility, recent studies have been performed to help identify
the target-cell type for AML. Hirouchi et al. (2011) gave 3 Gy to induce AML in mice. They
extracted marrow, sorted it using a range of different cell-type-specific markers, thentransplanted populations of HSC, MPP, CMP (common myeloid progenitors), CLP (common
lymphoid progenitors), B-cell/natural killer-cell, and T-cell types into normal mice. Bonemarrow was analysed in those grafted mice which developed AML. They concluded that (a)there was a possibility that hemizygous deletion of Dusp2 in chromosome 2 contributed tothe self-renewal potential of radiation-induced AML stem cells, and that (b) the initialradiation-induced AML stem-cell may originate not only from irradiated HSC but also from
MPP and CMP.
(A72) The plausibility of possible multiple target-cell types is further strengthened by the
recent work on induced genetic reprogramming of differentiated somatic cells into
embryonic-like stem cells (referred to as iPS cells). The latter appears to share many growthand maturation potentials seen in immortalised, pre-transformed (pre-cancerous) cells(Meissner et al., 2007). Also, supporting this suggestion are the older studies on retroviraltargeting of lineage-committed progenitors and the comparable “reprogramming” of virustargeted, lineage-committed haematopoietic progenitor cells into more primitive, stem-likeprecursor cells with enhanced, but often aberrant proliferation and differentiation potentials(Minucci et al., 2002).

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(A73) Lastly, as a contribution to the debate on leukaemogenesis mechanisms, a detailed
study was carried out regarding the “immortal strand hypothesis” (see section 2.3.5.) inhighly purified HSC, to detect if the parental DNA strand was retained during division cycles
to preserve its integrity (Kiel et al., 2007). They administered BrdU to newborn mice, micetreated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adultmice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs
retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs,
revealing that BrdU has poor specificity and poor sensitivity as an HSC marker. Sequential
administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs
segregate their chromosomes randomly. Division of individual HSCs in culture revealed noasymmetric segregation of the label. It was concluded that HSCs cannot be identified on thebasis of BrdU-label retention and do not retain older DNA strands during division, indicatingthat these are not general properties of stem cells.

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A.7. Summary and conclusions

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(A74) Radiation-induced leukaemias: Leukaemia is a rare, but very prominent disease of
the blood forming system. Although the a,y of most cases of leukaemia is unknown, arelatively small number of the diseases have been causally associated with prior exposures toa variety of physicochemical agents, most specifically ionising radiation and various classesof myelotoxic chemicals. Following extensive and detailed epidemiological evaluations ofleukaemia incidence within a Japanese cohort of A-bomb survivors, the AR of developing thedisease (in aggregate) is estimated at 2.7 x 10-4 per Sv per year, while risk estimates for the
major leukaemic subtypes- ALL, AML, or CML- are 0.6, 1.1, and 0.9 x 10-4 per Sv
respectively (Preston et al., 1994). CLL has not been consistently or convincingly increased
following exposure to ionising radiation.
(A75) Leukaemia, as a complex disease, is characterised by a highly aberrant,
dysfunctional blood-cell forming system of the body, a system that is normally highly
efficient and very reliable. This “cell producing factory” resides in the bone marrow andrelies heavily on the reproductive capacities of both primitive, and the not-so-primitive
progenitor cells. These cells appear to exist in a continuum of dual, reproductive capacities
that allows for balanced cycles of self-renewal and selective, lineage-specific commitment todifferentiate. It is a combination of the latter two cellular functions that become abnormal,
which is responsible for the disease.
(A76) Relevant data of various radiations and exposure types: In principal, ionisingradiation of all qualities, dose levels and conditions has leukaemogenic potential; however,disease risk can vary by orders of magnitude. Acute doses of whole-body exposures withdeeply-penetrating low-LET x- or γ−rays seemingly carry relatively high risks whencompared to exposures from select types of internalised, bone-seeking, high LET α-particleemitting radionuclides (e.g. 226Ra), in all likeliehood related to differences in the anatomical
distribution of dose in bone marrow.
(A77) General features of haematopoietic tissues: The marrow’s blood-forming capacity
is totally reliant on the well-orchestrated and coordinated activities of the major marrowcompartments: (1) stem cells and early haematopoietic progenitor cells; (2) proliferative,
lineage-committed daughter cells; and (3) non-proliferating maturing cells and associatedreserves. Tissues are bounded by encapsulating bone (spongy or compact), supported both
structurally and functionally by intra- and inter-stromal matrix, and both are vascularised andinnervated. The tissue construct is analogous to a tree in full foliage: the root system,
represented by the nutrient vasculature, with the endosteal matrix between enveloping bone

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and marrow and its mesenchymal osteo-haematopoietic precursor cells; the tree stem,
representing the supporting stem cell and early progenitor compartments; the rising major
branches, representing major cell lineages, in turn to a multitude of terminal branches,
representing amplified, maturing and finally, functional cells. Damage to the “stem”, i.e., thestem cell/progenitor compartment(s), will most certainly limit subsequent compartmental cell
amplifications, cell transitions, and maturation processes, and ultimately limit the capacity to
produce and to maintain circulating blood cell pools.
(A78) Being rather indiscriminate by nature (in terms of energy deposition), ionisingradiation will (if given the opportunity) produce damage along the entire haematopoietic
chain from primitive stem cell to the fully functional and mature blood cells. However, ingeneral, it is the stem cell/progenitor compartment(s) that is at pathological risk following
exposure.
(A79) Turnover rates: The estimated turnover rate for the bone marrow with the average
adult human is ~4.5 x 10-9 cells per day per kg of body weight (Fliedner, 1998). In humanmarrow, approximately two thirds of this activity is devoted to myelopoiesis, while theremaining third is dedicated to erythropoiesis. In total, this marrow activity serves to supply
and to renew on a daily basis circulating blood pools of ~2.5 x 109 erythrocytes, ~2.5 x 109
platelets, and ~10 x 109 granulocytes per kg body weight (Williams et al., 1990). The stem
cell compartment of the marrow comprises approximately 0.1 to 0.05% of the total tissue.
Precise turnover rates of stem cells and early progenitor cells within the marrow are uncertain
and remain to be determined. Nevertheless, rough estimates for marrow HSCs of several
species have been garnered via indirect methods: in humans, HSCs within marrow were
estimated to turn over once every ~45 weeks (Shepherd et al., 2004). Further, it is generally
accepted that the more primitive the species of stem cell/progenitor cell, the lower is its
turnover rate. Taking this to the extreme, it is quite possible that small numbers of marrowstem cells remain quiescent for the entire lifespan of the individual. This is another area of
debate that will require further work.
(A80) In terms of leukaemogenesis, a low turnover within virtually-quiescent marrowstem cells might well serve to reduce the number of potential, transformable cell targets forthe leukaemogenic action of ionising irradiation, regardless of LET or exposure intensity, byallowing time for repair of genomic damage prior to replicative transmission of that damage
to their daughter cells. This effect may be counteracted to an uncertain extent if somedaughter cells are also target cells.
(A81) Age dependence: Chronological age plays a significant role in radiation
leukaemogenesis. Preston et al. (1994) reported on the relative leukaemic risk within the Abomb survivor cohort, and found that with increasing age at the time of exposure, the risk
declined appreciably. In contrast, the estimated RR increased with time following exposure:in the very young survivor (0-9 years of age) sub-cohort, the risk steeply increased during theearly years following exposure; whereas in the older sub-cohorts, the rise in risk wassignificantly delayed and more gradual in slope. In general, animal-based studies support
these epidemiological findings.
(A82) Cellular features: A unique array of cell surface markers serves to distinguish
HSCs from other nucleated cells within bone marrow tissues. In the bone marrow of humans,
HSCs can be distinguished by the phenotype of CD34+, CD59+, Thy1+, CD38low/-, c-Kit-/low,and Lin-. The latter Lin- “marker” represents that absence of some 13 to 14 different surface
markers which characterise mature blood cells of the various blood cell lineages. By contrast,
HSCs of rodent (mouse) marrow, are distinguished by CD34low/-, SCA-1+, Thy1+/low, CD38+,
c-Kit+, and Lin- (NIH Report, 2008).

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(A83) The major subpopulations of HSCs, namely Lt-HSCs and St-HSCs, can be
distinguished via functional, time-based transplantation assays. In turn, the HSCs of various
marrow domains (e.g. endosteal, vascular) can be identified, as well as being distinguishedfrom multipotential HPCs by differential FACS analyses using SLAM, KDR, and otherrelated surface markers (Kiel et al., 2005; Ziegler et al., 1999).
(A84) Radiosensitivity: The radiosensitivity of HSCs and HPCs varies, and significantly
so, in ways which might seem counter institutive. The response (i.e. change inradiosensitivity with HSC/HPC maturation) appears “U” shaped, and not at all linear. The
most primitive of the HSCs (long-term marrow repopulating) are quite radioresistant,endowed with substantial SLDR and PLDR capacities, when compared to the moreradiosensitive CFU-Sday7 with selective lineage restriction, but still clearly multipotential by
nature. Like the MRAs, the more primitive CFU-Sday12 appears more resistant than the CFUSday7. A similar shift in radioresistance is noted as daughters of multipotential HPCs become
lineage-restricted and committed to specific differentiation lineages, e.g. granulopoiesis andassociated HPC transitions from GEMM-CFUs to GM-CFUs to M-CFUs.
(A85) Characteristics of single-cell responses: While it is true that the biotechnology ofmonitoring haematopoietic progenitor cells has significantly improved, still our inability toisolate from marrow, especially human marrow, the primitive HSCs to homogeneity in aconsistent and routine fashion, severely limits the application and utility of the more standard
biochemical and molecular approaches that are currently available. New cloning procedures
both in vitro and in vivo have been forthcoming, but still the results obtained from thesetechniques assume that the initial “seeding” cell is indeed the stem cell of interest and not amore mature progenitor cell which contaminates the seeding population.
(A86) Nevertheless, despite these technical limitations, progress has been made toward abetter understanding of the molecular and biochemical make-up of the more primitive blood
cell precursor cells found in the marrow. A good example of the latter progress is theapplication of differential genomic arrays in evaluating RNA transcript profiles of primitive,
pre-committed HSCs and finding low levels of transcription specific for multiple lineages.
This work has given rise to a new paradigm suggesting perhaps that lineage-commitment is abalanced, competitive process that involves not only the activation of lineage-specific genes,or sets of genes, but also, more importantly, shutting off activity of genes/gene sets,
associated with the unselected lineages. Another excellent example has been the use of
single-cell cloning of HSCs to identify and to characterise radiation-induced genetic
alterations that are passed down to daughter cells during early reproductive cycles.
(A87) Mutagenesis: Mutagenesis within the most primitive and rare HSCs remains illdefined, regardless of aetiology, due to the reasons mentioned above. The information that we
do have comes indirectly from the analyses of cloned daughter progenitor cells that are many
generations from the primary HSCs of interest. If it is assumed that the patterns andfrequencies of radiation-induced mutations are relatively constant for the different classes ofmarrow HSCs and HPCs, then the Hprt mutation analyses of in vivo (CFU-Sday11) and in vitro(CFUc) cloned marrow progenitors might be reflective of radiation mutagenesis within moreprimitive, long-term marrow-repopulating precursors. That is, the mutability of HSCs and
HPCs alike would be comparable to the mutability of progenitor cells from other body tissues,e.g. for Hprt mutations induced by low-LET x- or γ-rays, the mutation frequencies would bein the range of ~2 x 10-5 per Gy, while the spontaneous rates would be at least a factor of 10
lower.

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ANNEX B: MAMMARY GLAND STEM CELLS

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B.1. Radiation-induced breast cancer

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B.1.1. Radiation exposure and breast cancer risk: Epidemiology and physiology(B1) Breast cancer is the most commonly diagnosed cancer in women. Rates vary
between countries but in general the rate increases to age 50, approximately the age ofmenopause, after which the rate of increase is less. Together with the recognised contributionof life history, which includes age of menarche, age at first pregnancy, number and timing ofpregnancies, ovarian integrity, these data are indicative of the importance of ovarian
hormonal factors in breast cancer incidence.
(B2) Radiation exposure is a well-documented risk factor for breast cancer. The
UNSCEAR 2006 report (UNSCEAR, 2008) concluded that female breast tissue is moderately
radiosensitive, and that there is compelling evidence of excess cancer which increases
linearly with dose. There are complex modifying effects of age at exposure and attained age,as well as interactions with physiological processes, including pregnancy, and other risk
factors. Fourteen cohort studies provided quantitative information on the level of risk
following a wide range of doses in different populations around the world, as reviewed by
Boice (2001). Three key features were discussed: (1) linearity of dose response; (2) the
modification of risk by age at exposure, where the risk is inversely related to exposure age
and exposures past the menopausal ages appear to carry a reduced excess risk; and (3) the
effect of fractionating the dose on the subsequent risk is minimal. Although observed inindividual studies, a combined analysis of almost 78,000 women and 1,500 breast cancer
cases from eight cohorts confirmed linearity of dose response and little effect of dose
fractionation on AR and a decreasing risk with age at exposure (Preston et al., 2002).
Interestingly, a 6-fold decrease in risk was associated with protracted (as opposed to
fractionated) exposures compared with acute exposures in childhood.
(B3) Medical treatments are one source of radiation exposures linked to breast cancer.
Increased risk of breast cancer was noted in populations of girls with scoliosis, who wereusually monitored using x-rays from the onset of the growth spurt through puberty andadolescence (Boice Jr. et al., 1991). Tuberculosis patients who spanned a wide range of ages
also showed a greater breast cancer incidence later in life. The average dose to the breast was
lower in the scoliosis cohort (0.11 Sv) compared to two fluoroscopy cohorts (0.79 and 0.89
Sv). x-rays were used for treatment of benign breast disease (BBD) and acute postpartum
mastitis in women of child-bearing ages (Mattsson et al., 1993; Shore et al., 1986). Doses tothe breast were considerably higher compared to the above mentioned studies. Women with
BBD had a 3.58 fold increased risk for breast cancer when compared to a control group nevertreated with radiation (Mattsson et al., 1993). Women treated for postpartum mastitis who
were exposed to >1.5 Sv were at 2- to 4-fold increased risk of breast cancer compared to a
combined control group of siblings and unirradiated mastitis patients (Shore et al., 1986).
Radiation doses of several Gy used to treat haemangiomas on the chest region of children
under age 1 year were linked to elevated risks of breast cancer (Lundell et al., 1999).
(B4) Radiotherapy for breast cancer can result in a dose of several Gy in the contralateralbreast; radiation treatment for breast cancer was found to be related to development of
secondary breast cancers among women younger than 45 years of age (Boice et al., 1992;
Stovall et al., 2008). Land et al. (1994) conducted a nested case-control study of 196 breast
cancer patients and 566 matched controls among Japanese A-bomb survivors. Statistical101

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modelling indicated that early first birth, multiple births and long cumulative lactation history
protected not only against breast cancer per se, but also against radiation-induced breast
cancer in this population. The protective effect of age at first birth held for parous women at
exposure and for women who completed a first pregnancy following radiation exposure. The
protective nature of pregnancy may depend on additional factors. A pooled analysis of eightradiation-exposed cohorts showed that women with BBD or postpartum mastitis had thehighest excess rates of breast cancer following radiation therapy for those disorders, an effectmost pronounced among young women with BBD (Preston et al., 2002). The observation
might be partly related to the underlying association of BBD and breast cancer. The longlatency (approximately 40 years) between exposure and disease occurrence, the absence ofexcess when exposure occurs after age 45, and the effect of tissue and reproductive processes
suggest that breast biology is a strong determinant of radiation risk.
(B5) The rate of breast cancer in Japan is among the lowest in the world, but breast cancer
contributes a disproportionately large fraction of radiation-related cancer burden in A-bomb
survivors (Preston et al., 2007). The data from the Hiroshima and Nagasaki survivors provide
strong evidence for increased breast cancer following single acute doses of the order of 20
cGy and linearity of risk with increasing dose (Pawel et al., 2008; Pierce et al., 1996; Preston
et al., 2003b).
(B6) These studies also support the strong effect of age on modifying breast cancer risk, inthat there was no significant elevation among women exposed after the age of about 40 years
in 1945. Girls (approximately aged 10-14 years) exposed to ionising radiation at NagasakiHiroshima were much more likely to develop breast cancer than older girls or adult women
exposed to comparable radiation doses (Tokunaga et al., 1993). The risk was inversely related
to age at exposure, with the youngest children having the highest risk. The latency, that is thetime from exposure to development of breast cancer, was inversely related to age at exposure,i.e. the young children had to live until the ages later in life when breast cancer rates werehigh for the excess breast cancers to be manifest. A similar age effect was found for highdose radiation exposures to the breast from fluoroscopy for tuberculosis and radiation therapy
for Hodgkin’s disease (Boice Jr. et al., 1991; Hancock et al., 1993; Howe and McLaughlin,
1996).
(B7) More than 50,000 women in the US have been treated with chest radiation (≥20 Gy)
for paediatric or young adult cancers. According to the Childhood Cancer Survivor Study,breast cancer risk is greatest among women treated for Hodgkin's lymphoma (HD) with highdose mantle radiation, but it is also elevated among women who received moderate-dosechest radiation (e.g. mediastinal, lung) for other paediatric and young adult cancers, such as
non-Hodgkin’s lymphoma, Wilms tumour, leukaemia, bone cancer, neuroblastoma and soft
tissue sarcoma (Mertens et al., 2008). Children treated for cancer with radiotherapy have a
2.9 RR of subsequent malignancy compared to those without (Mertens et al., 2008). These
women are at significantly increased risk of breast cancer and breast cancer mortality
following cure of their primary malignancy. In a review of 11 retrospective studies and 3case-control studies (Henderson et al., 2010 ), the authors concluded that risk of breast cancer
increased as early as 8 years following chest radiation and did not plateau with increasing
length of follow-up. The authors point out that the cumulative incidence of breast cancer by
40–45 years of age ranged from 13–20%, and by 25–30 years of follow-up ranged from 12–26%. This incidence is similar to that in women with a BRCA gene mutation, where by age
40 the cumulative incidence ranges from 10–19% and is substantially higher than in young
women in the general population in whom the cumulative incidence of invasive breast cancer
by age 45 is only 1% (Satagopan et al., 2001). However, estimates of ERR 0.06 (95% CI:
0.01, 0.13) Gy-1 for HD survivors as a function of dose provides a lower ERR than that102

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estimated for the LSS, which is attributed to cell killing at high doses (Travis et al., 2003).
Moreover, those patients in which the ovaries receive ≥5 Gy, and are treated with certain
chemotherapies, have decreased risk due to ovarian deficit and early menopause, respectively.
(B8) A study from Milan of the molecular and marker analysis of the breast cancers of
women exposed to therapeutic radiation for childhood/young adult cancers, revealed a highrisk of breast cancer diagnosed at an early age (39 compared to 57 in a sporadic consecutive
series) and a higher frequency of developing estrogen receptor (ER)-negative tumours(Castiglioni et al., 2007). More than half (53%) of the breast carcinomas from irradiatedwomen showed features of basal-like tumours compared to 11% in a consecutive series ofbreast cancers not preceded by radiation. When compared to age-matched controls,
incidences of basal-like cancer were significantly higher (P = 0.0001) for women irradiatedafter breast maturation at the expense of human EGFR2 (HER2)-positive and luminal cancer
subtypes. Interestingly, there is little evidence of a disproportionate frequency of contralateral
ER-negative breast cancer in women treated with radiation for breast cancer, suggesting aphysiological basis for the shift identified in the Milan study. Consistent with this idea, thebreast cancer subtype in women treated with radiation after maturation was much more likely
to be ER- and progesterone (PR)-negative, p53 and cytokeratin (K) 5/6-positive, and lesslikely to be HER2+, compared to those whose radiation exposure occurred before maturation(Castiglioni et al., 2007). Additionally, Broeks and colleagues used expression profiling tocompare sporadic and radiation-preceded breast cancer. Unsupervised hierarchical clusteringof the profile data resulted in a clustering of 22 tumours obtained from patients whodeveloped breast cancer after HD compared to 20 control tumours. The radiation–preceded
tumours were characterised by genes associated with a more aggressive tumour type (Broeks
et al., 2010).
(B9) Both human and experimental animal data suggest that there are additional poorlyunderstood factors associated with ageing or other life events that are permissive of, or
actively promote, carcinogenesis. The preneoplastic phenotype in vivo is usually initiallyrecognised as a focus of autonomously proliferating cells that often exhibit surface antigen or
cytosolic enzymatic alterations. The subsequent appearance of neoplasia at the sites of suchlesions has been well-documented in experimental models in a variety of tissues (Farber,1987), but it is not clear what factors dictate the eventual development of clinical cancer. The
relatively restricted window of carcinogen susceptibility that is evident during or around
puberty in both rodents and humans has been postulated to either contain the greatest numberof target cells or be a critical period of stem cell regulation (Boice Jr., 2001). Based on theidea that stem cell transformation can lead to unlimited progeny, tissue-specific stem cells or
early progenitor cells are considered to be the critical cellular target in carcinogenesis as has
been discussed from many perspectives (Clifton et al., 1986; Potten and Loeffler, 1990; Reyaet al., 2001; Sell, 2004; Welm et al., 2002; Zeps et al., 1996).
B.1.2. Dose rate and radiation quality data
(B10) As noted above, total dose is associated with excess cancer, regardless offractionation, e.g. low radiation doses as occurred with repeated diagnostic fluoroscopy(Boice Jr. et al., 1991; Hoffman et al., 1989). In contrast to other organs like thyroid in whichrisk is greater for high dose rate than low dose rate, cancer risk for breast is different. Either
EAR or ERR for breast cancer risk in three high-dose-rate studies was not consistently
different of those from two dose-rate studies (UNSCEAR, 2008). Fluoroscopy studies
indicate breast sensitivity with no effect on lung cancer (Howe et al., 1995).

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(B11) In regards to radiation quality, there is little evidence that internal low-LET or highLET emitters are associated with excess breast cancer (UNSCEAR, 2006). Medical
exposures are photons in the range of 250 kVp, while the relatively high-energy γ-rays
produced by the A-bombs used in Hiroshima and Nagasaki would be less biologicallyeffective by a factor of 3. No elevation of breast cancer risk has been observed among several
studies of airline crews, who receive an average of 6 mSv/year, with 20-50% of the absorbed
dose attributed to the neutron component.

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B.2. General features of the mammary gland and carcinogenesis

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B.2.1. Tissue architecture(B12) The tissue architecture consists of the parenchyma that resides in a dense fibroblastic
stroma embedded in an adipocyte stroma. As in other tissues, the stroma and epithelial
components can be separated from each other using mechanical and enzymatic dissociation.
But unlike most other tissues, the epithelium can be destroyed and the stromal fat pad
maintained separately in vivo. The gland-free fat pad can become at any point the recipient oftransplanted epithelial cells that will repopulate the gland (DeOme et al., 1959). This featureprovided both unequivocal evidence of the MaSC, which has been a focus of investigation for
more than 60 years, and a functional assay for putative MaSC isolated from disaggregatedtissue.
(B13) The stroma plays an important role in determining tissue function. The embryonicmammary epithelial interaction with the dense fibroblast stroma determines the ability of theepithelia to interact with the fatty stroma (Sakakura et al., 1982). Rudimentary mammary
epithelium undergoes normal mammary morphogenesis in the mouse mammary fat pad offemales in the absence of reproductive hormones (Sakakura et al., 1979), but if the epithelium
does not come into contact with mesenchymal cells in the postnatal period, ductal
morphogenesis fails to occur (Kratochwil, 1969). Fetal mammary epithelial rudiments aresupported by adult stroma to fully differentiate (Sakakura et al., 1976). The tissue-specificpattern of ductal branching is dictated by stromal signals (Daniel et al., 1984; Sakakura et al.,1976). Sakakura and colleagues showed that heterotypic recombinants of fetal salivary
mesenchyme induce mammary epithelium to undergo ductal branching patterns typical ofsalivary gland, but at lactation, the isografts synthesised specific milk proteins (Sakakura etal., 1976). It was observed that the ductal-alveolar nodules formed by mammary epitheliumsalivary mesenchyme chimeras gave rise to a greater incidence of tumours (Sakakura et al.,1981). The mechanism by which perturbed tissue interactions contribute to the process ofneoplasia is not understood.
(B14) The processes of mammary gland development, differentiation and involution are
regulated by the ovarian hormones, estrogen and progesterone (Shyamala, 1997). Although
the mouse mammary gland is organised differently from the human breast, these generalfeatures are similar and both are ovarian hormone dependent (Shyamala, 1997). Studies inmice have shown that estrogen provides the primary stimulus for ductal morphogenesis
during puberty while concomitantly priming the gland for lobular-alveolar differentiation via
its induction of PR. Progesterone is subsequently necessary for elaboration of the ductal tree
via side-branching in the virgin animal and alveolar expansion during pregnancy (Atwood etal., 2000). While estrogen and progesterone are critical for proliferation, it is clear thatmammary epithelial cells differ in their ability to respond to these signals. During both ductal
and lobular-alveolar mammary growth, the distribution of proliferating cells is heterogeneous,104

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suggesting the involvement of local factors in dictating the specific response to systemic
hormones (Bresciani, 1968; Christov et al., 1993; Daniel et al., 1987).
(B15) Estrogen signalling is mediated initially by ERα or β, members of the nuclear
receptor superfamily of transcription factors. The precise roles of these receptors in
regulating mammary development are complex (Shyamala et al., 2002). Both receptors
impact on mammary carcinogenesis and are expressed in a subset of tumours and hence, also
impact on their therapeutics. However, ERβ is dispensable for mammary development in
mice, while ERα is required. This will be discussed in detail because the ER status of breast
cancer is an important prognostic feature, a critical therapeutic target, and the ER status ofbreast cancer has been shown to be affected by radiation in both humans and mice
(Castiglioni et al., 2007; Nguyen et al., 2011).
(B16) Studies in adult human breast indicate that ER expressing luminal epithelial cells also
express PR, and constitute 20-30% of the epithelium (Clarke et al., 1997b). The frequency ofERα cells increases with age in human breast paralleling the rise in breast cancer risk (Shoker
et al., 1999). Lawson et al. (1999) proposed that an increased frequency of cells increases
breast cancer risk. Women at higher risk of breast cancer have more ERα cells compared withthose women in a low risk population. Japanese women living in Hawaii had more ERα thanthose in Japan, which parallels cancer risk (Lawson et al., 2002). ERα cells are increased innormal tissue of tumour-bearing breasts (Khan et al., 1994), in postmenopausal women(Shoker et al., 1999), and in postmenopausal women using hormone replacement therapy(Lawson et al., 2001).
(B17) Anderson and colleagues showed that in contrast to the uterus, ERα does not colocalise with DNA synthesis or with broad markers of proliferation in human breast (Clarke
et al., 1997b). Additional studies have confirmed this in human and in normal mouse and rat
mammary glands (Ewan et al., 2005; Russo and Russo, 1998; Saji et al., 2000; Turgeon et al.,
2001; Zeps et al., 1998). Nonetheless, proliferation of ER+ cells does occur, albeit at a low
frequency that is highly restricted. The overall frequency of ERα cells co-localised withmarkers of proliferation was 0.2% in the study from Anderson and colleagues (Clarke et al.,1997b). Russo et al. (1999) showed that the distribution of proliferating ERα cells depended
on the type of lobule in human breast, and demonstrated an inverse relationship between the
degree of human breast lobular differentiation and co-localisation. The least-differentiatedtype 1 lobule averaged 0.48% co-localisation of Ki67 and ERα, while the more-differentiatedtype 3 lobule exhibited only 0.01 % dual labelling. Proliferating ERα cells are thought torepresent a distinct lineage compared to non-proliferative cells. Dual-labelled cells are
significantly more numerous in all proliferative lesions compared to normal pre- or postmenopausal lobules and correlate positively with increasing level of breast cancer risk
(Shoker et al., 1999), but curiously do not occur frequently in breast cancers themselves(Jensen et al., 2001).
(B18) Mouse models have been used to determine whether ERα cells cannot or do not
proliferate, i.e. are they incapable or restrained from responding to signals? Mammary
epithelial proliferation is increased approximately 4-fold in adult TGFβ1+/- mice, at oestrus(Ewan et al., 2002). The majority of the cells were ER-negative, indicating that decreased
exposure to TGFβ permits cells to more readily respond to hormone-mediated signal. Notably,
TGFβ1+/- mice also exhibit a significant increase in cells dual-labelled for ER and Ki67 or
BrdU (1 hour labelling) compared to wild-type mice at oestrus (Ewan et al., 2005). Thus, atleast some ER cells are capable of proliferating, supporting the idea that they represent aprogenitor population.
(B19) Studies in rats also showed that radiation exposure followed by pregnancy reduced
tumourigenesis (Clifton et al., 1975). It was speculated that differentiation of a given
105

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mammary cell for milk secretion markedly reduced the proliferative potentiality of that cell.
In other words, in the presence of hormones that stimulate end-differentiation of the
mammary cells, irradiation-altered mammary cells are lost from the high proliferativepotential population. Along these lines, Sivaraman et al. (2001) proposed a cell-fatehypothesis to explain the protective effect of pregnancy on breast cancer risk in general. Theysuggested that the hormonal milieu of pregnancy affects the developmental fate of a subset of
mammary epithelial cells, perhaps by altering signal transduction and/or gene expression,
which thereby reduces the risk of breast tumour development. This hypothesis would support
the data from A-bomb survivors showing that early pregnancy reduces the risk of radiationinduced breast cancer.
B.2.2. Features of mammary stem cells in situ
(B20) It is now generally accepted that "stemness" is not a single property, but a number ofproperties that can be manifested under different conditions (Booth et al., 2000). As stated byPotten, a stem cell must be undifferentiated (relative to other epithelial cell types, but notnecessarily relative to embryonic cells) and capable of proliferation, self-maintenance, andregeneration of the tissue after injury (Potten and Loeffler, 1990). It must be capable ofproducing many differentiated progeny, and retaining the ability to switch between these
options when appropriate. These properties also make stem cells an important target forneoplastic transformation.
(B21) Great advances have been made in the first decade of this century in isolating
putative MaSCs from rodent and human tissues in laboratories around the world. As
mentioned above, the demonstration that mouse mammary epithelial cells could seriallyrepopulate the mammary gland provided the first evidence that the mammary gland contains
a stem cell (Daniel and Deome, 1965). It is widely believed that understanding the biology of
MaSC in human breast and rodent mammary gland is fundamental to understanding thedevelopment of cancer. However it is now well appreciated that the properties, and probably
the number, of stem cells may change in response to circumstances, including experimental
manipulations.
(B22) Experiments by Smith and colleagues first demonstrated the clonal origin of murine
mammary outgrowths (Kordon and Smith, 1998). Mammary epithelial cells were marked byrandom insertion of mouse mammary tumour virus and serially transplanted to show that
outgrowths arose from a single cell based on the pattern of viral insertion sites. Large cell
patches with the same blueprint X-chromosome inactivation in the human breast support theexistence of a stem cell (Tsai et al., 1996). Terminal ductal-lobular units, the structures thatcap the ducts in the human gland, are mono-phenotypic regarding X-chromosome
inactivation (Diallo et al., 2001), as also occurs at the smaller ducts (Preston et al., 2003c).
Interestingly, both luminal cells lining the ducts and surrounding myoepithelial cells present
matching patterns of inactivation, suggesting that they are derived from a common precursor.
(B23) The stem cell distribution is restricted within the architecture of many adult tissues,
like the crypt of the small intestine (Potten and Loeffler, 1990) or the hair follicle inepidermis (Jensen et al., 1999). But samples taken from any portion of the mammary gland at
any age and at any developmental stage, including full functional differentiation, give rise to
mammary epithelial outgrowths with complete developmental capacity. Smith and Medina(1988) proposed morphological features which can be used to distinguish MaSC populations
in mouse, rat and human mammary glands. Cytological examination of mouse mammary
gland explants revealed the presence of morphologically distinct cells distributed sporadicallyamong the mammary epithelium, whose behaviour in vivo and in vitro suggested that they106

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might represent a latent epithelial stem cell population. The authors of these studies suggested
that rare small cells that possess relatively few cytoplasmic organelles and dispersed
chromatin are undifferentiated MaSCs. Using osmium tetroxide staining and electron
microscopy, Chepko and colleagues identified small light cells (SLCs), undifferentiated large
light cells (ULLCs), and an intermediate population between SLCs and ULLCs, which theysuggested, might indicate that SLCs differentiate into ULLCs (Smith and Chepko, 2001).
Both SLCs and ULLCs are division-competent, as evidenced by the presence of mitotic
chromosomes (Chepko and Smith, 1997). Interestingly, both cell types can appear as single
cells, but are also found as homogeneous pairs (two SLCs or two ULLCs), suggestive of
symmetric division, or in mixed pairs formed by one SLC and one ULLC, suggestive ofasymmetric cell division. SLCs are suprabasal and do not have contact with the lumen, while
ULLCs can appear in contact or not with luminal areas.
(B24) The observation that SLCs constitute a constant fraction of the mammary epithelium
through pregnancy, lactation and involution, suggests that this population increases anddecreases as necessary, indicative of a stem-like behaviour for these cells (Chepko and Smith,1997). However, the frequency of these cells (about 3% of the epithelium) is too large for a
MaSC population, thus suggesting that a committed progenitor cell also might be included in
the SLC pool. Division competence, staining properties and the presence of mixed pairs of
SLCs and ULLCs make these latter cells a candidate progenitor cell population. TheseULLCs were postulated then to give rise to both secretory and myoepithelial cells. Notably,both SLCs and ULLCs are depleted in growth-senescent tissue (Smith et al., 2002), i.e.
mammary outgrowths from a third-generation transplant or beyond that are unable to giverise to further mammary tissue upon transplantation into a cleared fat pad. In contrast, SLCsand ULLCs are readily detectable in hyperplastic alveolar outgrowths, which can bepropagated indefinitely by serial transplantation, which was suggested to be consistent with a
MaSC-like population. Moreover, rat SLCs seem to be surrounded by a niche, formed bydifferentiated luminal cells and myoepithelial cells, as well as stretches of direct contact withthe basement membrane (Chepko and Dickson, 2003). These niches contain 1 to 4 SLCs,
with one of them always close to the basement membrane. While conceptually attractive,these studies lack functional tests that SLCs or ULLCs are stem cells since preparation of thetissue for electron microscopy eliminates the possibility of either isolating or staining forother markers (e.g. hormone receptors).
(B25) A functional assay of long-lived populations, so-called LRCs, has been used in
mammary glands (Kenney et al., 2001; Welm et al., 2002; Zeps et al., 1996). Depending on
physiological status, stem cells are generally thought to be quiescent during normal tissue
homeostasis (Cheng et al., 2000; Lowry et al., 2005; McCroskery et al., 2003). 3H-thymidine,
BrdU or a fluorescent cell tracker can be used to follow proliferation and persistence of cells
in which label was incorporated. The label is provided for a short period (e.g. 2 weeks)during which the majority of cells undergo DNA synthesis. A wash-out period of several
weeks is then observed during which the label is either diluted beyond detection, which isapproximately 5 divisions, or the cell is lost. Cells that retain label many weeks (e.g. 8-9)
after the initial labelling period are considered to be early progenitors based on the model inwhich stem cells and progenitor cells divide infrequently (Potten and Loeffler, 1990).
(B26) LRCs have been previously studied in the mouse mammary gland, although theprotocols used to identify these cells have been different every time. When mature mice werelabelled by injecting them with 3H-thymidine, different distributions in the number of cells
that incorporated and retained the label over a two-week chase were observed depending of
the stage of the oestrus cycle at the time of label injection (Zeps et al., 1996). The distributionof label intensity was also variable. This is due to the fact that different hormonal levels
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during the oestrus cycle regulate the size of the proliferating compartment in the mammary
gland. However, in all cases, the most heavily labelled cells were at some distance from theclosest cells with similar degrees of label retention. This would point towards the presence of
discrete units randomly distributed along the mammary tree containing one stem cell and its
closest progeny. Most LRCs thus identified had a luminal location and expressed the ER(Zeps et al., 1998), although a subpopulation of heavily labelled, ER- basal cells was also
described. Interestingly, the frequency of these cells is more consistent with that of a stem
cell population, and their basal location agrees with some other reports which indicate that
the MaSCs have a basal (Deugnier et al., 2002) or suprabasal (Chepko and Smith, 1997)
location. The fact that the initial labelling was done on adult, non-stimulated mice, indicatesthat most mammary MaSC, quiescent during tissue homeostasis, should not haveincorporated the label and therefore, would not be included in the LRC population identified
in these studies.
(B27) LRCs seemed to be located into discrete, periodical units along the mammary ducts
in mammary tissue transplants following BrdU administration in drinking water and with an
8-week ‘chase’ (Kenney et al., 2001). These units likely contain both stem cells and theirearliest progeny. LRCs did not express markers of milk production, like whey acidic protein(WAP) or β-casein, and a subpopulation of them did not express adhesion molecules, like αcatenin and zonula occludens 1 (ZO-1). This might be related to the maintenance of thesecells in a niche. Inexplicably, some ducts seemed not to possess any LRCs.
(B28) BrdU can also be delivered using micro-osmotic pumps that release their content at
a constant rate (Theeuwes and Yum, 1976). Using this approach, it was possible to label 3week-old mice (entering puberty, where the mammary MaSC pool is supposed to divide
symmetrically) for two weeks (Welm et al., 2002). This was then followed by a 9-week‘chase’. This approach presents several advantages. First, BrdU release is continuous rather
than in pulses. Second, the stage in the oestrus cycle of the mice did not influence the results,
since both labelling and chase periods are relatively long and include several complete cycles.
After the chase, very few LRC were found that expressed the progesterone receptor. A
subpopulation not expressing luminal or myoepithelial markers was also described within the
LRC compartment. Finally, when the SP of these mice was analysed, LRCs were shown to befour times more abundant in the SP than in the remaining cells.
(B29) An alternative interpretation of LRCs proposed by Cairns is that stem cells maintaindifferential strand segregation that protects the stem cell DNA from replication errors (Potten
et al., 2002). Double-labelling experiments using 3H-thymidine and BrdU provide support forthis mechanism in mouse mammary gland (Smith, 2005). The tissue was first labelled withradioactive thymidine after transplantation into cleared fat pads, which mimics mammary
development, when MaSCs are supposed to divide symmetrically. After a chase period, BrdU
coupled with a proliferative stimulus (hormonal treatment, which should induce asymmetric
MaSC division) was used to further label the dividing cells. Most epithelial LRCs
incorporated the second label, thus showing that, although quiescent, they have a highproliferative potential. Stromal LRCs did not seem to be able to incorporate the second label,
but this might have been due to an inadequate proliferative stimulus. When a second chasewas carried out, most LRCs lost the second label, but not the first label. This indicates a
selective segregation of DNA in these cells in agreement with the Cairns hypothesis, andtherefore validates the use of LRCs in the mouse mammary gland, as long as the initiallabelling is done during tissue development (normal or post-transplantation).
(B30) Transplants of human breast epithelium into athymic mice have recently been used
to study human LRCs (Clarke et al., 2005). Most LRCs were shown to be out of cycle (theyexpressed p27, a cyclin-dependent kinase inhibitor) and enriched for expression of p21 and108

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Musashi 1 (Msi-1), both putative stem cell markers. However, these two markers are
expressed in distinct subfractions of the LRC pool, with p21+ LRCs in suprabasal locations
and Msi-1+ LRCs in luminal or suprabasal positions. This might indicate the presence of two
different stem cell compartments with different potentials, or a stem cell hierarchy where amultipotent stem cell gives rise to a unipotent stem cell. Interestingly, most of the cells
expressing p21 or Msi-1 in the outgrowths were hormone receptor positive.
(B31) An advantage of LRC techniques is information in situ unavailable withdisaggregation approaches like cell sorting profiles or mammosphere-forming cells. A
systematic characterisation using image analysis techniques, together with the classic
immunofluorescence and microscopy approaches, has catalogued the properties, frequency
and distribution of LRCs (Fernandez-Gonzalez et al., 2010; Fernandez-Gonzalez et al., 2009).
Immunofluorescence was used to identify LRCs that incorporated BrdU while undergoing
DNA synthesis during puberty (3-5 weeks of age) and retained this label in the adult gland(14-18 weeks), as well as epithelial cells expressing PR and a transcription factor p63,
indicative of specific functions. Approximately 4% of luminal cells were LRCs, the majorityof which did not express PR, a luminal epithelial differentiation marker, indicative of
undifferentiated luminal cells. Multi-scale analysis in situ, which is high-content image
analysis measurements of multiple features (e.g. population size and distribution, localorganisation or cellular properties) linked at multiple scales (e.g. organ, tissue, single cell),revealed that luminal LRCs have a distinct nuclear morphology, are enriched 3.4-fold in large
ducts, and are distributed asymmetrically across the tissue. A population of suprabasal cells,
located between the luminal and the myoepithelial layers, was highly enriched for LRCs.
Myoepithelial cells express p63 and represent a differentiated population. Many suprabasal
LRCs exhibited both the luminal LRC nuclear morphology and the myoepithelial marker p63,
which suggests that they have bi-lineage differentiation potential. Together, these datasuggested that the ventral-most, large ducts contain a reservoir of MaSCs. In support of this,CD24 and CD49f epithelial cells were enriched in preparations from the ventral versus thedorsal gland.
(B32) Lineage tracing is used to experimentally test whether a marker is truly expressed instem or progenitor cells. An example is that of an orphan 7-transmembrane receptor, Lgr5,
which was identified using lineage tracing as a marker of cycling stem cells in the gut (Barker
et al., 2007). This transgenic technology creates a mouse in which the expression of a reporter
protein, often fluorescent, is transiently driven by the promoter of the protein in question tomark all the progeny of the cell expressing the designated marker at the time of induction.
For example, Lgr5, which is a Wnt target gene, also marks stem cell compartments of theskin and stomach. However, in the mammary gland, the progeny of Lgr5+ cells does not markstem cells per se but marks lineage specific cells (de Visser et al., 2012). Interestingly, Lgr5
was shown to mark luminal cells immediately after birth but switches to myoepithelial cells
at 12 days postnatal. Adding to this complexity is cytokeratin lineage tracing experimentsfrom Blanplain that suggest there are restricted basal and luminal stem cell compartments inthe mouse mammary gland (Van Keymeulen et al., 2011).
B.2.3. Turnover rate and age dependence(B33) Cell turnover in the mammary gland is a response to a complex life cycle dictatedby systemic factors. The mammary gland is established via mesenchymal induction ofectoderm specialisation. At birth, the gland consists of an epithelial anlagen that is mainly
quiescent until proliferation and morphogenesis are stimulated by the hormones of puberty.
Proliferation within the epithelium varies from highly proliferative endbuds to relative
109

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quiescence among the epithelium of the nipple. Short periods of proliferation occur withoestrus in mammals, followed by apoptosis which maintains the ductal tree throughout life.
Ovariectomy leads to regression and an essential static state. Pregnancy induces considerable
expansion (i.e. from ~10% epithelium to 90%) accompanied by secretory differentiation. The
period of lactation is sustained by suckling and can persist for years in humans. Cessation ofsuckling leads to rapid apoptotic involution and tissue remodelling to a quasi-nulliparous
state.
(B34) Whether the unusual age dependence of radiation breast cancer risk is linked tophysiological processes unique to mammary development remains widely debated. An
estimate of the number of stem cells (clonogens) in the mammary gland was first reported by
Clifton and Gould. They reported on the successful takes and extent of growth of seriallydiluted mammary epithelial cells into the interscapular fat pad of rats, and estimated thatmammary epithelial clonogens were present at a frequency of approximately 0.05% in thecell population of the virgin rat mammary gland (Gould et al., 1977). Experiments performedin mice have demonstrated that any portion of the mammary tree (i.e. primary duct, tertiaryduct), any developmental stage (i.e. virgin, lactating), or any age (i.e. 3 weeks, 80 weeks)contains cells capable of repopulating the mammary stroma and undergoing the complete
developmental cycle of the parenchyma (Neville and Daniel, 1987). These experiments
convincingly demonstrated that totipotent stem cells exist throughout the mammary
parenchyma tree and are not localised to just the terminal portions of the mammary tree.
(B35) The number of the clonogenic cell subpopulation increases during ductal
morphogenesis during puberty, and is apparently stable subsequently despite cycles ofdifferentiation and involution. Rapid and extensive proliferation during pregnancy is
primarily that of TA cells that will undergo secretory differentiation, followed by apoptosis
during involution upon cessation of suckling. There are some experimental data suggestingthat stem cells expand with hormonal stimulation (Joshi et al., 2010), but this remains
controversial due to interpretation of functional assays. Involution returns the gland to a state
similar to that of the nulliparous animal, but with distinct changes that are transiently
associated with increased risk of cancer but confer long-term protection, at least if occurringin young women (Schedin, 2006). Notably, the repopulation potential of the epithelium, as
evidenced by serial transplantation of the mouse mammary gland, is maintained into old age,
but the capacity of the host to support mammary gland outgrowth decreases with age (Daniel
et al., 1968). Thus, the increase of stem cells in adults compared to puberty appears to beroughly inversely proportional to susceptibility to carcinogenesis.
B.2.4. Cellular features: stem cell markers(B36) Many markers have been used to enrich or identify stem cells but none aredefinitive in the mammary gland or breast epithelium at the time of this publication.
Cytokeratins, lineage commitment antigens and stem cells markers from other tissues havebeen used to characterise MaSCs. Thus, for example, keratin 6 (K6), which is very rarely
expressed in mouse luminal epithelial cells, has been postulated as a MaSC marker (Smith
and Chepko, 2001). This is based on two features: (1) in both mouse and human cultures,
only colonies formed from luminal cells display multiple phenotypes (Pechoux et al., 1999;
Smalley et al., 1999); and (2) K6 is expressed in the proliferative area of primary cultures ofmammary epithelium (Smith and Chepko, 2001). Thus, it is likely that the MaSC population
resides in the luminal epithelium, and that since K6 seems to mark cells with highproliferative potential, the rare luminal epithelial K6+ cells might represent a stem cell

110

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population. However, no functional characterisation of this population has been successful atproving enrichment in stemness potential.
(B37) A population of suprabasal, luminal cells was isolated from human mammary tissue
based on the expression of epithelial cell surface antigen (ESA, a luminal marker expressed at
the basolateral surface of the cells) and the absence of muchin 1 (MUC1) expression (anapically-expressed luminal marker) (Gudjonsson et al., 2002). These cells were thenimmortalised to generate a suprabasal cell line, which was able to generate both luminal(expressing K18) and myoepithelial (expressing K14) cells in clonal assays. The suprabasal
cell line was formed by cells that also expressed K19, known to be restricted to a luminal
subpopulation in situ. Interestingly, in clonal cultures, this cell line also showed multipotency
regarding K19 expression in its progeny. However, no hormone-receptor positive cells wereobtained in vitro. These data seem to indicate that the suprabasal cell line contains not a stem,
but a progenitor, cell population. However, functional characterisation of these cells bytransplantation into cleared mouse fat pads might shed light on the hormone sensitivity of theresulting outgrowths. Noted above is that basal and luminal cytokeratins appear to markdistinct lineages in the mouse (Van Keymeulen et al., 2011), which may point to a species
difference.
(B38) Luminal and myoepithelial lineage markers, epithelial membrane antigen (EMA)
and common acute lymphoblastic leukaemia antigen (CALLA), respectively, seemingly
expressed later in the commitment process than cytokeratins (see above), have been used to
identify candidate stem cell populations in the human breast (Clayton et al., 2004).
Interestingly, populations that are either double positive (0.47% of the epithelium) or double
negative (30%) for both of these markers were able to generate mixed (luminal andmyoepithelial) colonies, but double negative cells generated more colonies with a luminal
phenotype. Most double negative cells expressed K18, a luminal marker. This might indicatethat double negative cells contain a luminal progenitor population. In that case, themyoepithelial colonies generated by these double negative cells would confirm the
hypothesis of a myoepithelial progenitor within the luminal compartment [see above,(Gudjonsson et al., 2002)]. Only double positive cells showed proliferation markers (very few
cells) or expressed ER (at low levels). When these cells were grown in the presence of
estrogen, they generated fewer luminal colonies, thus suggesting a role for estrogen in cellfate decision. However, the loss of ER expression after two days in culture (Kothari et al.,2003), together with other potential changes in gene expression that the cells undergo in vitro,
prevent drawing any definitive conclusions from these results regarding the role of ER in
regulation of cell commitment. In any case, both the double positive and double negative
populations seem to contain progenitors with some degree of commitment, as shown byexpression of ER (double positive cells) or K18 (double negative cells).
B.2.5. Functional analysis of mammary stem cells(B39) Functional analysis of stem cells has been the gold standard for HSCs using bonemarrow repopulation following lethal irradiation. Likewise, MaSCs can be demonstrated bytheir high repopulation potential. A series of break-through experiments was carried out in
the Cancer Research Lab at Berkeley in the 1950s, where mouse mammary epithelial cells
were transplanted into inguinal mammary fat pads from which the epithelial compartment
had been excised (so-called cleared fat pads) (DeOme et al., 1959). Transplanted tissue or
dissociated cells proliferate, undergo ductal morphogenesis to fill the fat pad, and pregnancy
initiates differentiation to produce milk. The age of the donor does not have an effect on theoutgrowth potential of the transplant (Young et al., 1971). These experiments clearly showed111

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that there must be a pool of cells within the mammary epithelium with extensive regenerative
potential. Furthermore, the fact that serial transplantation experiments can be made up to fourto six serial iterations with similar efficiency (Daniel et al., 1968) indicates that these cells
not only can give rise to a fully functional mammary gland, but also are able to self-renew
many times. Similar properties have been shown for rats (Gould et al., 1977; Kamiya et al.,
1998; Kim et al., 2000) and for human mammary epithelial cells transplanted into mouse fatpads (Clarke et al., 1997a; Laidlaw et al., 1995).
(B40) The rational for the SP sorting scheme for stem cells is based on the hypothesis thatstem cells are resistant to chemical damage to their DNA because they express ATP-binding
cassette (ABC) transporter family proteins which are able to “pump out” certain chemical
products (Jonker et al., 2005; Zhou et al., 2002). One of these products is Hoechst 33342.
Using flow cytometry on cells counterstained with this dye, a subpopulation of cells that haveexcluded it from their nuclei, the SP can be identified. In the bone marrow, this (SP)subpopulation has been shown to be highly enriched for HSCs (Goodell et al., 1996; Goodell
et al., 1997).
(B41) A mouse mammary SP also has been identified (Welm et al., 2002). This population,
which constitutes about 2-3% of the epithelium, is formed by relatively small cells, and isenriched for expression of Sca-1, a stem cell marker in the haematopoietic system (although
it is also broadly expressed in the mammary epithelium). The mammary SP is also enrichedfor LRCs (see below), which might be indicative of a quiescent phenotype in situ.
Transplantation experiments revealed that approximately 1/4000 SP cells can give rise to afunctional outgrowth in a cleared mammary fat pad. However, the cells had to be cultured for
3-5 days before sorting and transplantation. This step, which might have induced celltransformation (e.g. by enhancing the ability of cells to efflux the dye (Alvi et al., 2003)), wasnecessary to increase the cell viability through Hoechst staining and sorting. Interestingly, ina different study (Alvi et al., 2003), a mouse mammary SP was isolated without intervening
cell culture, and a much smaller fraction of cells (0.45% of the epithelium) was obtained.
(B42) A human SP, constituting 0.18% of the epithelium, was likewise isolated in thatlatter study (Alvi et al., 2003). These cells have comparable expression profiles to non-SP
cells regarding luminal (K18) and myoepithelial (K14) markers. However, when different
luminal (EMA) and myoepithelial (CALLA) markers were assayed (Clarke et al., 2005;
Clayton et al., 2004), the human SP cells were shown to be mostly negative for both of them.
This might indicate that the different lineage markers used are expressed at different stages ofthe differentiation process, with keratins being “turned on” before EMA or CALLA. When
assayed for colony formation, single human mammary SP cells were able to form colonies
expressing K18, K14 and both markers, thus suggesting a multilineage potential for thesecells. Self-renewal of the SP has also been shown by the fact that virtually all the
mammosphere-forming cells (see below) are included in the SP in human tissue (Dontu et al.,2003).
(B43) Contradictory results regarding ER expression in these cells have been reported(Alvi et al., 2003; Clarke et al., 2005; Clayton et al., 2004), and it is therefore difficult toconclude whether they are sensitive or not to the mitogenic stimulus provided by estrogen. SP
cells seem to be enriched for cells expressing telomerase (Alvi et al., 2003), as well as otherpotential stem cell markers, like p21 or Msi-1 (Clarke et al., 2005). Also, activation of theWnt pathway (involved in stem cell self-renewal (Reya and Clevers, 2005)) increases the sizeof the mouse mammary SP both in primary cultures and in transgenic mice (Liu et al., 2004).
All this evidence, although sometimes contradictory, points towards the existence of a stem(or early progenitor) subpopulation within the SP compartment. However, SP identification

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requires destruction of the tissue microenvironment, thus rendering impossible the study ofthese cells within their tissue context.
(B44) Sca-1, a MaSC marker in the haematopoietic system, has also been used to isolate apopulation of luminal, mouse mammary epithelial cells with enriched regenerative potential
upon transplantation into cleared fat pads (Welm et al., 2002). Sca-1+ cells are very frequentin the SP (75% positive cells versus 20% in the entire epithelium), and they contain twice as
many LRCs as the Sca-1- fraction. When labelled in situ, Sca-1+ cells do not express PR,
another steroid hormone-related protein, or differentiation markers. However, they constituteabout 20-30% of the epithelium, which makes unlikely that Sca-1 labels a stem-cell-only
population, but a progenitor or TA cell population.
(B45) Along these lines, it has been shown that a population of mouse mammary epithelial
cells, where 1 in 20 cells is able to repopulate a fat pad, expresses low levels of Sca-1 (Stingl
et al., 2006). These cells were sorted based on their high expression of CD24, a neural MaSC
marker, and CD49f (α6-integrin), which labels epidermal MaSCs. Gene expression analysis
showed that these cells have very similar profiles to myoepithelial cells, with no significant
differences: most of them were positive for smooth muscle actin or K14, a few were positive
for K18, and none expressed both markers. Also, very few of these cells expressed K6,
previously postulated as a MaSC marker (Smith and Chepko, 2001). The SP contained lessthan 10% of these cells (Stingl et al., 2006), thus indicating that the SP may represent moredifferentiated progenitor cells, because its repopulation efficiency was not as high as it wasfor the CD24+ CD49fhigh population. CD49f is expressed as a heterodimer with CD29 (β1integrin). In a very systematic study using CD24 and CD29 expression (Shackleton et al.,2006), a similar enrichment for regenerative potential in the CD24+ CD29high population (1 in64) was shown, and this confirmed the keratin expression patterns observed previously. The
CD24+ CD29high epithelial cells had increased colony-forming potential in two-dimensional(2D) cell cultures, while in three-dimentional (3D) culture they gave rise to both ductal andalveolar structures with luminal and myoepithelial populations. This study demonstrated forthe first time, regeneration of a fully functional outgrowth after transplantation of a single
CD24+ CD29high cell into a cleared fat pad (with and without support cells). The outgrowths
obtained recapitulated the CD24/CD29 expression profile of intact glands. All these pieces ofdata demonstrate the multilineage potential of the CD24+ CD29high cells. When cells from oneof these outgrowths (generated from a single CD24+ CD29high cell) were transplanted into
multiple fat pads, a functional outgrowth was obtained in each of the recipient fat pads. Thisshows that the CD24+ CD29high cell that gave rise to the outgrowth must have self-renewed.
Therefore, this study demonstrates that the CD24+ CD29high population in the mouse
mammary epithelium is highly enriched for cells with multilineage and self-renewal
potentials, the two properties that define a MaSC. Interestingly, these cells are enriched for
LRC content, but not for Sca-1 expression or Hoechst exclusion (SP), and they do not form
mammospheres.
(B46) Transcriptional profiling of FACS-sorted CD44high CD24low breast epithelial stem
cells demonstrated an enrichment in transcripts determining cell motility, cell adhesion, cellproliferation, chemotaxis and angiogenesis. A striking observation was the enrichment intranscripts for TGFβ and Wnt signalling components in these stem cells (Shipitsin et al.,2007). Indeed, the stem cell compartment was responsive to TGFβ, and targeted by TGFβ
inhibition, whereas CD44low CD24high progenitor cells had lost responsiveness due todownregulation of the TGFBR2 gene. Clinical evidence demonstrated that expression of a
“TGFβ cassette” of genes (expressed in CD44high CD24low > CD44low CD24high) in breast
tumours is associated with a shorter metastasis-free survival of breast cancer patients with
ERα-negative tumours (Shipitsin et al., 2007). The transcriptional similarity between normal
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and neoplastic stem cells was greater than that between stem cells and their CD44low CD24highprogeny within the same tissue. It would appear that, as in embryonic stem cells, TGFβ
signalling plays a role in cancer stem cell maintenance.
(B47) Taking this observation one step further, it was shown that inducing ‘Snail’overexpression leads to epithelial-mesenchymal transition or transformation (EMT) in humanmammary epithelial cells that then exhibit stem-cell-like properties in terms of expression ofstem cell markers, increased mammosphere seeding activity in vitro and tumourigenicity invivo (Mani et al., 2008). TGFβ frequently mediates the transcriptional EMT programme(Zavadil and Bottinger, 2005; Zavadil et al., 2004). It is noteworthy that radiation exposure
induces TGFβ activation in mouse mammary gland (Barcellos-Hoff, 1993, 1994), and that
irradiated human mammary epithelial cells activate TGFβ and are predisposed to undergo
TGFβ mediated EMT (Andarawewa et al., 2007). Excessive TGFβ levels in the irradiatedmicroenvironment may therefore not only maintain stem cells, but also contribute to theirformation via EMT of more differentiated progenitors. This possibility remains to be tested.
(B48) An important caveat to these experimental studies is that most of studies make useof cell sorting techniques that require disruption of the tissue microenvironment before
obtaining a stem cell-enriched population. Most of the antibodies used in flow cytometry
analysis have ligands on the cell membrane or in the cytosol. When tissue sections were
stained for CD24+ CD29high, it was hard to identify cells that met the criteria due to the broad
expression of these markers in situ coupled with the compactness of epithelial tissues(Shackleton et al., 2006). In ducts, CD24+ CD29high cells seemed to be basolateral, while inend buds (the growing portion of the ducts during gland development) expression was higher
in cap cells, the invading cell layer, supposed to be a stem cell reservoir (Williams and Daniel,1983). Thus, although CD24+ CD29high can be immunostained in vitro (where cells are nicely
isolated or form relatively small clumps), when these markers are stained in situ, large
populations of positive cells are identified. Furthermore, it is often difficult to tell what cell ispositive and which one is not, especially when dealing with cell surface labels. This prevents
the use of these markers for studies of the in situ organisation of stem-like populations andtheir microenvironment.
(B49) The frequency of mammary repopulating cells isolated from dissociated mammary
gland is generally assumed to accurately reflect the frequency and behaviour of MaSC in situ,
i.e. the presumption is that stemness is a property inherent to certain cells rather than being
conferred by the microenvironment or context. The recent identification of heterogeneous
populations with mammary repopulating properties, yet lacking multipotent lineage
commitment evidence in situ, together with the struggle to obtain pure MaSC populations,suggests that both cell intrinsic and extrinsic properties are important in stem cell behaviour.
This underscores the need to identify the niche that harbours stem cells in order to understandand control stem cells themselves. As of now, identification of the indisputable mammary
MaSC niche has been elusive.

4738	
  

B.3. Radiosensitivity

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B.3.1. Stem cell and progenitor cell radiosensitivity(B50) Few studies have attempted to assess the radiation sensitivity of the MaSCs orprogenitor cells. A comprehensive analysis of the radiosensitivity of rat mammary clonogens
has been conducted by Gould and colleagues (Kamiya et al., 1990; Kamiya et al., 1998;
Kamiya et al., 1991). In this model, monodispersed rat mammary epithelial cells are injected
into the intrascarpal fat pad where they form clonogenic organoids. The surviving fraction of114

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clonogenic mammary cells was measured in groups of virgin rats from 1 to 12 weeks after
birth after single exposures to 137Cs γ rays (Shimada et al., 1994). The radiosensitivity ofclonogens from prepubertal rats was high and changed with the onset of puberty at between 4
and 6 weeks of age. Additional studies suggest that the ability of immature tissue to process
chemical carcinogen damage is different form that of the mature tissue (Ariazi et al., 2005).
(B51) Studies to test the hypothesis that mammary progenitor cells are resistant toradiation suggest that the relative resistance of a progenitor population is mediated at least inpart by Wnt signalling, which is implicated in stem cell survival (Woodward et al., 2007).
Freshly isolated mammary epithelial cells following in vivo radiation (4 Gy) and sorted by
FACS for cells similar to that published (Shackleton et al., 2006), did not enrich for this stem
cell population and in contrast, decreased this population by approximately one-third.
However, radiation selectively increased the Sca-1+ population in vivo, the SP of primarymammary epithelial cells after 3 days of culture, and the Sca-1+ fraction of MCF7 breast
cancer cells and COMMA-D murine epithelial cells. The authors concluded that mammary
progenitor cells are relatively resistant to radiation, which they attribute to increased survivin
signalling (Chen et al., 2007; Woodward et al., 2007).
B.3.2. Single cell responses(B52) When irradiated clonogenic mammary epithelial cells are transplanted andhormonally stimulated, they give rise to clonal glandular structures within which carcinomas
may arise (Gould et al., 1991). Using this experimental strategy in breast and thyroid, Cliftonand colleagues found frequencies as high as one cancer per 10-300 clonogens (Kamiya et al.,1995). Similarly, Kennedy and Little found similarly high initiation of transformation in
irradiated C3H10T1/2 mouse fibroblasts (Kennedy et al., 1980; Kennedy and Little, 1978).
Both epithelial and fibroblast events were suppressed by high density or addition of
unirradiated cells. These data and others led to the conclusion that initiation can be a highfrequency epigenetic event rather than a low frequency mutational process (Kamiya et al.,1995).
(B53) Growth in suspension culture systems has been known to be a property of cancer
cells (Freedman and Shin, 1974) but is also attributed to stem cells. Dissociated humanmammary epithelial cells cultured on a non-adherent substratum undergo anoikis, i.e. death
due to the lack of anchorage. A few cells survive, however (Soule and McGrath, 1986), andunder specific culture conditions they give rise to multicellular spheroids, which in the caseof breast are called mammospheres (Dontu et al., 2003). To demonstrate stem cell activity,
dissociated mammosphere cells were shown to give rise to luminal, myoepithelial andalveolar cells in monolayer culture (seeded on tissue culture plastic at clonogenic densities),
and to complex, branching, ductal-alveolar systems in 3D culture conditions (Dontu et al.,2003). Furthermore, when mammospheres are dissociated into single cells and grown again
in suspension, they can form new mammospheres with multilineage potential, which can be
repeated indefinitely. The more passages, the more bipotent progenitors (measured by thefrequency of mixed luminal and epithelial colonies obtained in monolayer) are present in the
mammospheres. Successive passaging does not alter the frequency of mammosphere
formation or their size, indicating that all the spheroids arise from cells with self-renewal
competence. This shows that mammospheres contain cells with multilineage and self-renewal
potentials, the two properties expected of a stem cell population.
(B54) This approach allowed protein expression and transcriptional profiling studies inorder to identify human MaSC markers (Dontu et al., 2003). For example, CD10, α6-integrin
or K5 were all shown to be expressed by cells in the centre (and not in the periphery) of115

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mammospheres. Transcriptional profiling showed that mammosphere cells shared
upregulation of many genes with ES, NSC and HSC. For example, the Notch pathway has
been involved in cell fate decision (Artavanis-Tsakonas et al., 1999). Using the
mammosphere system, it was shown that treatment with an inhibitor of Notch signalling
reduced the size of primary mammospheres, and inhibited formation of secondaries (Dontu etal., 2004). On the other hand, addition of an activator of that same pathway induced anincrease in the frequency of mammospheres with each passage, and also with respect tountreated controls, suggesting an increased number of mammosphere-forming cells in eachgeneration due to activation of self-renewal. These Notch-activated mammospheres also
contain an enlarged fraction of myoepithelial progenitors as shown by the increased
frequency of mixed and myoepithelial colonies obtained in monolayer cultures ofmammosphere-dissociated cells. This indicates a role for Notch in inducing proliferation ofbi-potent and myoepithelial progenitor cells. However, this mammosphere-forming potential
cannot be identified in situ, and therefore, as with the SP, it is not possible to study thelocation and arrangement of these cells within the mammary gland or the potential presenceof a niche around them.

4811	
  

B.4. Experimental models of carcinogenesis

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B.4.1. Initiation(B55) Evidence from rodent models indicates that neoplastic initiation by radiation or
chemical carcinogen is relatively frequent (Clifton et al., 1986), while progression toneoplasia is relatively less frequent (Medina et al., 1986). Studies from Gould and Clifton
showed that one in approximately 13 irradiated (7 Gy) rat mammary clonogens gave rise tocancer, which indicated that at least 1 of approximately 95 clonogens was radiogenicallyinitiated (Kamiya et al., 1995). A similar high initiation frequency was seen in grafts ofmethylnitrosourea (MNU)-treated clonogens. Such initiation is thus far more frequent thanspecific locus mutations. In sites grafted with larger cell inocula, cancer incidence perclonogen was suppressed inversely as the number of irradiated or MNU-treated clonogens per
graft increased. Addition of unirradiated cells to small irradiated graft inoculates also
suppressed progression. In a variety of systems, the expression of dysplasia in vivo andneoplastic transformation in culture was inversely correlated with the number of cells seeded(Clifton et al., 1986; DeOme et al., 1978; Ethier and Ullrich, 1982; Terzaghi-Howe, 1990;
Terzaghi and Nettesheim, 1979). The nature of the cell dose dependence is unclear but
appears to be due to suppression of the initiated cells’ expression of its altered phenotype by
the normal cells. These epithelial-epithelial interactions are clearly involved in a variety ofnormal and neoplastic processes.
B.4.2. Progression(B56) In mouse mammary gland, hyperplastic nodule formation is one distinct
morphological marker of preneoplasia (Banerjee et al., 1987). Depending on the carcinogen,parous status and hormonal environment of the gland, such hyperplasias will appear to be
ductal,alveolaror
ductal-alveolar.
Somecarcinogens,such
as7,12dimethylbenz(a)anthracene (DMBA) and radiation, produce ductal hyperplasia and dysplasia(Ethier and Ullrich, 1982), which are similar in their range of severity and histology to thoseabnormalities observed in the pathogenesis of human cancer (Ethier et al., 1984). As withmouse mammary tumour virus (MMTV) lesions (DeOme et al., 1978), dissociation and116

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transplantation of exposed cells into host glands increase the expression of lesions over thoseobserved in intact mammary glands, indicating that the potential for expressing preneoplastic
lesions is present long before they are expressed in the intact gland (Ethier and Ullrich, 1982).
The size of the inocula and the age of the animal at analysis also affect the frequency andseverity of the dysplasia (Ethier et al., 1984). Interestingly, a 10-fold smaller inoculum ismost effective. This is thought to reflect the increased number of cell divisions required to fill
the mammary gland, which allows for a greater opportunity to express the altered phenotype,but also could be due to a suppressive action of more non-transformed cells (Kamiya et al.,1995).
(B57) Analysis of the mammary outgrowths from irradiated cells showed that analysis ofan actively growing (immature) or in a resting state (mature) gland allows a distinction to bemade between the expression of the preneoplastic phenotype, which is greatest in the growing
gland, and whether the altered phenotype persists in the resting gland (Ethier and Cundiff,
1987). Some lesions regress or remodel following the cessation of growth. However, thealtered phenotype could be re-expressed upon retransplantation even in an apparently normal
recombinant gland. Persistence of a lesion following cessation of gland growth is considered
to reflect the acquisition of the ductal dysplasia for some autonomy from tissue regulatory
mechanisms. Increasing the time between carcinogen exposure and transplantation increased
the frequency of persistent preneoplastic lesions, which also indicates progression of cells
that continue to acquire new characteristics with time. If a year is allowed to elapse between
exposure and transplantation then a third characteristic is observed: the ability to elicit a host
response (Adams et al., 1987). The dynamics of neoplastic progression were found to be
similar for exposure to DMBA or radiation at these low doses, and this reveals commonalities
between two qualitatively different carcinogens. However, DMBA did induce more persistentlesions than radiation, although when 1 year elapsed between exposure and transplantation,
this difference was eliminated.
(B58) A qualitative relationship between preneoplastic lesions and tumourigenesis is
generally accepted, because such lesions have been noted to precede neoplasia andprogressively acquire phenotypes consistent with increasingly autonomous behaviour.
However the quantitative relationship is less well understood. The efficiency of tumour
induction is usually related to dose of a given agent, but other factors such as the toxicity of
the agent or the hormonal status of the tissue can influence tumour incidence. For example, at
DMBA exposures below 0.125 mg, tumour incidence is highly dose-dependent, at doses
above 0.25 mg, the toxic and carcinogenic effects in other tissues become important, and at
higher doses, the histological tumour type changes from ADC to adenocanthomas (Ethier and
Ullrich, 1982). The preneoplastic lesions preceding such dose effects have not been studied.
Experimental models that maximise the progression to frank tumours for radiation have beendeveloped by altering the hormonal environment of the gland (Clifton et al., 1986) but have
not yet been studied in terms of their specific effects on preneoplastic lesions.
(B59) As discussed above, Ullrich developed a novel assay to show that radiationinfluences progression using mammary cells dissociated from mice 24 hours to 16 weeks
after being irradiated and subsequently injected into gland-free fat pads to assess thefrequency and characteristics of ductal dysplasias in outgrowths (Ethier and Cundiff, 1987;
Ullrich, 1986). Notably, this assay suggests that the acquisition of the altered growthpotential which resulted in ductal dysplasias and the ability of these lesions to gain someautonomy from growth-regulatory mechanisms were separate events that occurred at different
times after carcinogen treatment. Radiation quality and dose rate affect both characteristics.
For example, mice irradiated with fission-spectrum neutrons at dose rates of 1 cGy/min or 1
cGy/day were assessed (Ullrich, 1986). Dysplasias from cells irradiated at high or low dose
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rates were similar, but a large fraction of the dysplasias in outgrowths derived from mice
irradiated at low dose rate persisted, whether collected at 24 hours or 16 weeks, while thosefrom mice irradiated with a high dose rate did not persist. These data suggest that low-doserate neutron exposures enhance the probability of progression of carcinogen-altered cells
rather than increase the number of initiated cells.
B.4.3. Tumourigenesis(B60) The efficiency of radiation-induced tumourigenesis depends on dose and the qualityof radiation (Fry et al., 1983). Rats stimulated with high prolactin levels and glucocorticoid
deficiency (Prl+/Glc-) for 48 days increased 3H-thymidine uptake by nearly 4-fold, and total
mammary clonogens by about 5-fold (Kamiya et al., 1999). Irradiation with 4, 40, and 80
cGy x-rays after hormone stimulation increased total mammary carcinomas per rat-day-atrisk linearly with dose, while a dose of 40 cGy x-rays before hormone treatment yielded
tumour frequencies insignificantly different from unirradiated controls. In contrast to resultswith x-rays, 10 cGy neutrons prior to hormone treatment yielded tumour frequencies andlatencies insignificantly different from those for 10 cGy neutrons after hormones. The authors
concluded that the carcinogenic action of x-rays, but not of neutrons, was thus influenced bytotal clonogen numbers and/or their proliferation rates.
(B61) A series of studies in rodents demonstrates the age, dose and radiation quality
dependence of mammary carcinogenesis. When 7–8-week-old ACI, F344, Wistar, andSprague-Dawley rats were evaluated until 1 year of age after irradiation (0.05–2 Gy) witheither 290 MeV/u carbon ions with a spread-out Bragg peak (LET 40-90 keV/µm) generated
from the Heavy-Ion Medical Accelerator in Chiba compared to 137Cs γ rays. Carbon ionssignificantly induced mammary carcinomas in Sprague-Dawley rats but less so in otherstrains. The dose-effect relationship for carcinoma incidence in the Sprague-Dawley rats
provided an RBE of 2 at high dose per fraction but was estimated to be 10 at low doses(Imaoka et al., 2007). Metastasis was noted only in animals exposed to high LET radiation.
(B62) Recent studies from Shimada and colleagues support the concept that physiologicalstatus is critical factor in determining the subsequent cancer risk (Imaoka et al., 2011).
Female Sprague-Dawley rats irradiated at a dose of 0.2 or 1 Gy with either 137Cs γ rays or a
290-MeV/u monoenergetic carbon ions (LET 13 keV/µm) at embryonic days 3, 13, and 17 or
15 weeks after birth did not develop more tumours over a lifetime (90 weeks) compared with
the non-irradiated group. However, among the groups of rats irradiated 1, 3 and 7 weeks after
birth, similar dose responses (0.2-2.0 Gy) to γ rays were evident. Moreover, the effect ofcarbon ions increased along with the age at the time of irradiation, indicating RBE values of
0.2 (-0.3, 0.7), 1.3 (1.0, 1.6) and 2.8 (1.8, 3.9) (mean and 95% CI) for animals that were 1, 3,
and 7 weeks of age, respectively.
B.4.4. Microenvironmental influences(B63) The stroma is also a possible target of radiation effects. Epithelial-stromalinteractions, mediated by the extracellular matrix, play a pivotal role in normal mammary
gland biology (Bissell and Aggeler, 1987). Normal stroma can suppress neoplastic
progression (Cooper and Pinkus, 1977; Decosse et al., 1973; Fujii et al., 1982; Kamiya et al.,1995). Specific stromas are induced by breast cancer (Finak et al., 2008). Indeed it has been
postulated that cancer can be promoted by an abnormal stroma (reviewed in (Barcellos-Hoff,
1998; Bissell, 2001; Farber, 1984; Tlsty, 1998)).

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(B64) Barcellos-Hoff developed a mammary chimera model to test whether radiation
effects on the stromal microenvironment contribute to its carcinogenic potential (BarcellosHoff and Ravani, 2000; Nguyen et al., 2011). This model consists of surgically removing theendogenous epithelium of the mammary gland at puberty, the mouse irradiated and the gland
subsequently transplanted orthotopically with unirradiated, non-malignant epithelial cells. In
one set of experiments, host irradiation with a high dose (4 Gy) up to two weeks before
transplantation with unirradiated, immortalised mammary epithelial cells, led to rapid
formation of large, aggressive tumours even though normal outgrowths formed in nonirradiated hosts (Barcellos-Hoff and Ravani, 2000).
(B65) There are several other examples of such ‘non-targeted’ radiation effect. A series ofexperiments from the laboratory of Kaplan in the late 1950’s showed that mice irradiated (~7Gy) and then transplanted with an unirradiated thymus develop thymic lymphoma at the same
incidence and latency as intact irradiated mice (Kaplan et al., 1956). An immortal myogenic
cell line forms tumours rapidly in irradiated mice but gives rise to normal tissue in
unirradiated host muscle (Morgan et al., 2002). Likewise, brain tumours develop in Ptch
mutant mice following partial body irradiation (3 Gy) even when the brain is shielded(Mancuso et al., 2008). Together, these studies are evidence for mechanisms of radiation
acting via persistent changes in the host or microenvironment.
(B66) A recent study used the radiation chimera model consisting of donor epithelium
primed to undergo neoplastic transformation by genetic loss of p53 transplanted to mice
irradiated with a low dose (<0.1 Gy) (Nguyen et al., 2011). Syngeneic p53 null mammary
gland transplanted to wild-type hosts develops normal ductal outgrowths but undergoes ahigh frequency of spontaneous transformation. This is evident histologically around 8 months
post transplantation as ductal carcinoma in situ and genomic instability. By 12 months, most(~60-70%) transplanted glands exhibit palpable tumours that are classified as carcinomas(Medina et al., 2002). In radiation-chimera experiments in which mice were exposed to dosesof 0.1-1 Gy, the first tumours were detected at about 170 days post transplantation in both
irradiated and non-irradiated hosts. By 300 days, 100% of transplants in hosts irradiated witheither 0.1 or 1 Gy had developed tumours compared to 54% of transplants in unirradiatedhosts. Tumour development was significantly accelerated by more than two months in hosts
exposed to low dose irradiation. Surprisingly, host irradiation significantly increased
development of ER-negative tumours. The effect of host irradiation on ER-negative tumour
frequency was not associated with the effect of radiation on latency per se.
(B67) ER is perhaps the most important clinical marker in breast cancer and is associatedwith distinct risk factors, pathological features and clinical behaviour (Jensen et al., 2003),
but what determines the prevalence of ER negative cancer is not well-understood (Allred andMedina, 2008). ER negative breast cancer is most frequent in young women and certain racial
groups, particularly African-American women (Parise et al., 2010). A single study by
Castiglioni et al. (2007) revealed that tumours from women exposed to therapeutic radiation
for childhood/young adult cancers were more likely to be ER-negative, specifically triplenegative breast cancer, compared to age-matched controls. Broeks et al. (2010) reported thatgene expression profiles of radiation-preceded breast cancer were consistent with basal-like
intrinsic subtypes, more aggressive, and could be clustered compared to those occurring in
women diagnosed at the same age without radiation treatment. Shimada and colleagues
reported that irradiation of Sprague-Dawley rats at 3 weeks of age produced carcinomas thatwere negative for either ER or PR, whereas the majority of carcinomas arising in rats
irradiated at 7 weeks (post puberty) were positive for both ER and PR (Imaoka et al., 2011).
The observation from Nguyen et al. that irradiated hosts were significantly more likely togive rise to ER- and PR-negative tumours implicates radiation-induced heterotypic signalling
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in determining critical clinical features of breast cancer. These data in both experimental
models and humans suggest that radiation can not only increase the risk of cancer, but also
ultimately affect the type of cancer.
(B68) It has been proposed that breast cancer heterogeneity is determined in part by thecell of origin and its position within the epithelial lineage hierarchy of normal organs (Sell,
2004). A corollary is that the tumours retain fundamental programming that remains evident
in the biology, behaviour, and signature of the cancer subtype. Indeed, the expression profilesof isolated MaSCs, which are thought to give rise to luminal progenitor cells that in turngenerate mature luminal cells, segregate breast cancers with specific markers and prognoses(Lim et al., 2010). Mouse p53 null tumours are similar to claudin-low breast cancer (Prat etal., 2010) and both are enriched in the MaSC signature (Lim et al., 2010). Moreover,neoplastic transformation in p53 null mammary gland is thought to be enhanced by apropensity for stem cell self-renewal (Cicalese et al., 2009), which is mediated by Notch
signalling (Li and Marban, 2010). Notch is preferentially activated in the normal ductal
luminal epithelium and promotes commitment of MaSCs in vivo (Bouras et al., 2008). Notch
nuclear co-localisation was significantly increased by radiation (Nguyen et al., 2011). Thus,
radiation could affect stem cell activity by inducing key regulators of mammary self-renewal
and lineage commitment. Gene expression profiling was carried out to evaluate the signalling
pathways that mediate the communication between cell types after radiation exposure. The
tumours arising in the irradiated host have a strong MaSC profile, as does the irradiatedmammary gland (Nguyen et al., 2011).
(B69) Together, these data suggested the hypothesis that low dose host irradiation might
affect the mammary lineage hierarchy by altering self-renewal in MaSCs. To test this idea,
mice were irradiated with graded low doses at 3 weeks of age, and cells isolated from fully
mature glands were analysed by FACS using CD24medium CD49high mammary repopulation
markers (Shackleton et al., 2006). The proportion of Lin- CD24medium CD49high cells in
irradiated mice was significantly increased compared to sham-irradiated mice. The absence ofdose dependence indicates that this effect is not mediated by cell kill per se. Functional
analysis of repopulating potential is the gold standard to assess MaSCs (Purton and Scadden,2007). Mammary repopulating activity increased nearly 2-fold in mice irradiated at 3 weeks
of age compared to sham-irradiated mice, again without evidence of dose dependence
between 0.1 and 1 Gy. Thus, low doses of ionising radiation induce a tumour promoting
microenvironment by induction of Notch pathway that in turn increases MaSC activity, andcorrelates with the increased frequency of ER-negative tumours (Arvold et al., 2011).

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B.5. Summary and conclusions

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(B70) The most striking feature of breast cancer risk following radiation, as compared toother organs, is the strong and consistent age-at-exposure effect (Boice Jr., 2001). Why isthere a window of susceptibility during adolescence? Six fundamental alterations in cell
physiology underlie cancer progression: self-support in growth signals; insensitivity to
growth-inhibitory signals; escape from apoptosis; infinite replication; sustained angiogenesis;
and tissue invasion and metastasis (Hanahan and Weinberg, 2000). These same underlying
mechanisms that contribute to cancer progression affect cell processes needed for normal
mammary development. The biology of breast differs in that the parenchyma is generated
post-natal, primarily due to action of ovarian hormones whose action is initiated with puberty.
The nascent epithelial bud expands to generate an adult ductal tree that is capable ofpreparatory cycles of proliferation as a function of the menstrual cycle, and explosive
proliferation and differentiation under the hormones of pregnancy. The capacity of the organ

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to undergo repeated expansion, differentiation and involution throughout adult life speaks toa huge reservoir of regenerative capacity. This capacity is thought to reside in MaSCs whose
self-renewal during puberty leads to their distribution throughout the gland.
(B71) Weissman and colleagues have argued that understanding stem cell biology canprovide insight into the origins of cancer (Reya et al., 2001). They propose that thesimilarities between self-renewal in stem cells and cancer cells could be exploitedtherapeutically if more was known about the MaSC populations. Low doses of radiation
significantly increased the mammary repopulating activity, and could thereby increase thenumber of target cells that could initiate cancer (Nguyen et al., 2011). Understanding theeffect of radiation on the MaSC is likely to provide key insights into physiological andgenetic determinants of cancer risk. The data in the radiation-chimera mammary model(Nguyen et al., 2011) suggest that radiation exposure early in life can alter heterotypicinteractions, set the stage for stem cell expansion and increase the risk of developing ERnegative breast cancer observed in women treated with radiation for childhood cancers(Castiglioni et al., 2007). A plausible scenario is that radiation elicits a transient change insignalling or a persistent change in the inflammatory, macrophage, or vasculaturecompartment of the gland. This altered microenvironment in turn permanently alters the poolof mammary epithelial stem/progenitor cells.
(B72) Moreover, gene expression profiling of radiation-preceded tumours is a new avenue
for exploring the effects of radiation both on the source cells and host biology. Mao et al.
(2005) suggested the hypothesis that specific early events after radiation exposure inducechanges in caretaker genes, and the nature of the early events may determine the overall
genomic signature observed in the resulting tumour. Consistent with this, analysis of theexpression profiles of radiation-preceded breast cancer implied that a large number of thesegenes are indeed caretakers and gatekeepers (Broeks et al., 2010). The radiation-chimera
mouse model suggested that host irradiation alone can ‘imprint’ the tumour with the MaSC
signature and shift the biology toward a more aggressive phenotype (Nguyen et al., 2011).
This ‘imprint’ or metaprofile can actually cluster radiation-preceded thyroid cancers fromspontaneous cancers (Nguyen et al., 2013).
(B73) Stem cell biology has thus provided important insights both in the biology of breast
cancer and a growing body of evidence that radiation may affect breast cancer risk by alteringtissue composition and stem cell regulation. The rapidly advancing knowledge of criticalpathways, as well as definitive stem cell markers in breast in humans and mammary gland inexperimental models, will likely provide greater insight into the age dependence of breast
cancer risk in humans.

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ANNEX C: THYROID STEM CELLS

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C.1. Radiation-induced thyroid carcinogenesis

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(C1) Ionising radiation is a well-established causative agent for thyroid cancer, especiallypapillary thyroid carcinoma (PTC), in humans. The relationship between external irradiationand late occurrence of thyroid cancer was reported originally by Duffy and Fitzgerald (1950).
Thyroid cancer was the first solid tumour that showed significant increases among A-bomb
survivors (Thompson et al., 1994; Wood JW, 1969). Pooled analytical studies of paediatricpatients exposed to external radiotherapy demonstrated a strong positive association between
radiation exposure and thyroid carcinoma (Ron et al., 2012). Based on the ERR model, alinear dose-response relationship fitted the data well (Ron et al., 2012; Shore, 1992). The
elevated risk among aromic bomb survivors has persisted for over several decades after the
initial exposure. It began to decline after 30 years, but was still elevated at >50 years(Furukawa et al., 2013; Ron et al., 2012).
(C2) An age-dependent susceptibility to external radiation of the thyroid gland exists. The
increased incidence of thyroid cancer was observed among the A-bomb survivors under 20
years of age at exposure (Furukawa et al., 2013; Thompson et al., 1994), whereas the elevatedrisk was no longer observed among persons exposed after the age of 30 years. Recent
analysis using data on cancer incidence during 1958-2005 demonstrated that persons exposed
early in life had a very high risk of thyroid cancer (Furukawa et al., 2013; Preston et al.,2007). In the latest A-bomb data (Furukawa et al., 2012), the ERR was estimated to decrease
4.5-fold between ages 10 and 30 at exposure and yet further at older ages. Although one
analyst reported that thyroid cancer incidence among females exposed at over the age of 20
was dose-related in the A-bomb study (Richardson, 2009), the most recent data indicate little
or no effect from adult exposures. Similarly, a pooled analysis of studies of external radiation
exposure and subsequent thyroid cancer reported that there was “little risk apparent after 20
years” (Ron et al., 2012). The RR tended to be greater in women, but the difference between
men and women was not statistically significant.
(C3) On 26 April 1986, the nuclear reactor No. 4 at the Chernobyl nuclear power plantreleased approximately 1,760 PBq of 131I into the environment. More than 90% of the thyroid
dose was attributed to ingested 131I, while less than 10% was from short-lived isotopes, suchas 132I, 133I and 135I. Individual thyroid doses ranged from a few milligrays to several grays(Ron 2007). A few years after the accident, an increase of thyroid cancer among children in
the contaminated area was reported (Prisyazhiuk A, 1991). Subsequently, several studies
revealed a substantial increase in thyroid cancer incidence among children exposed in thethree affected countries (Abelin et al., 1994; Baverstock et al., 1992; Likhtarev IA, 1995;
Stsjazhko et al., 1995; Tsyb et al., 1994). A recent report on a large population-based casecontrol study included 276 children with thyroid cancer diagnosed between 1992 and 1998
versus 1,300 matched controls (Cardis, 2005). A linear dose-response relationship wasobserved up to 2 Gy, and an estimated ERR was approximately 4.5 per Gy. The study also
demonstrated that the risks were higher in children in iodine-deficient areas. The first cohort
study of thyroid cancer following the Chernobyl accident screened 13,127 people in Ukraine
who were younger than 18 years of age at the time of the accident. A strong andapproximately linear dose-response relationship was demonstrated, which yielded an
estimated ERR of 5.25 per Gy (Tronko et al., 2006) for thyroid cancer prevalence.
Subsequent rounds of screening found an ERR for thyroid cancer incidence of 1.91 per Gy122

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(Brenner et al., 2011). A similar dose response was reported from a screening of 11,970
people in Belarus with an ERR per Gy of 2.15 (Zablotska et al., 2011).
10

	
  

9
Incidence per 100,000 8	  Children (0-14 yr)	
   Adolescents (15-19 yr)	
   Young adults (20-21 yr)

	
   	
  

7

	
  

6
5
4
3
2
1
0

	
  
	
  	
  
	
  

	
  

	
  
	
  

	
  

	
   	
  
	
  

	
  
	
   	
   	
  
	
  
	
  
	
   	
  	
   	
   	
  
	
  
	
   	
  
	
   	
  
	
   	
   	
   	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  
	
  	
   	
  	
   	
   	
  	
   	
  
	
   	
   	
  
	
  
	
   	
   	
   	
   	
  
	
  

86
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90

95

00

05

Calendar years

Fig. C.1. Incidence of thyroid cancer in different age groups in Belarus between 1986 and 2006
(Demidchik, 2007). (Permission needed)(C4) The annual incidence of thyroid cancer, examined in patients of different age groups
in Belarus, documented a striking increase in the incidence of thyroid cancers. In children(aged 0-14 years), the incidence of thyroid cancers started increasing 3-4 years after 131I
exposure in the presence of population screening (Demidchik, 2007). The cohorts exposed to131
I in childhood or adolescence continued to show excess risk of thyroid cancer as they
progressed through adolescence and their 20’s and early 30’s, whereas those born after 1986
showed no excess thyroid cancer risk.
(C5) Development of thyroid carcinomas after an injection of 131I as well as external
exposure to x-rays, was also demonstrated in experimental animals (Goldberg and Chaikoff,
1951; Doniach, 1956; Lee et al., 1982). Both internal irradiation with 131I (0.925-1.48 MBq)and external x rradiation (5-10 Gy) resulted in destructive lesions in the thyroid gland, which
in turn induced sequential changes from focal regenerative hyperplasia to benign nodule
formation (Lindsay and Chaikoff 1964). Higher doses of 131I such as 7.4 or 14.8 MBq, or 20
Gy of x-rays, almost completely destroyed the thyroid gland, preventing epithelial
regeneration (Frantz, 1957; Field, 1959). As the focal hyperplasia and benign nodule
formation were dependent on thyroid stimulating hormone (TSH), TSH has been considered
to be a promoting factor in development of thyroid carcinoma (Hall, 1948). Cardis et al.
(2005) reported that thyroid cancer risk was 3 times higher in areas with deficient dietaryiodine. While α-particle radiation was reported to cause degenerative lesions in the thyroid
gland (Hamilton, 1950), its carcinogenic effect is unclear.

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C.2. Human data on radiation qualities and type of exposure

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(C6) Internal radiation exposure from 131I used for diagnostic testing and therapeutic
treatments in adults showed no increase in the risk of thyroid cancer, while therapeutic use of131
I in adults with hyperthyroidism suggested a small effect regarding thyroid carcinogenesis

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(Hall and Holm 1998; Schneider and Sarne 2005). Prior to the Chernobyl accident, an excess
risk of thyroid cancer was observed among children living in the Marshall Islands, where
only a small contribution was from 131I (Schneider and Ron, 2000). A study of the children
exposed to nuclear fallout from nuclear weapons at the Nevada Test Site demonstrated asmall albeit statistically insiginificant excess of thyroid cancer (Kerber, 1993; Lyon et al.,2006). Data from the study of childhood exposure to radioactive iodine released from the
Hanford Nuclear site showed no evidence for an increased risk of thyroid neoplasms (Davis,
2004), while a recent report claimed that the results could be due to inadequate statisticalpower and proposed to interpret the results as inconclusive (Hoffman, 2007).
(C7) Although a pooled analysis has shown that external irradiation with thyroid doses of
0.5 Gy or more increase the risk of human thyroid carcinoma (Ron et al., 1995), there weresome studies examining the doses of the order of 0.1 Gy (Ron 1989, Pottern 1990, Shore
1992). The effects of fractionated external radiation were also examined in nuclear workers,
radiologists and radiologic technologists (Polednak 1986, Wang 1990, Boice, 1992, Zabel,2006). Some associations between occupational exposure and thyroid cancer risk wereobserved; however, the results need confirmation with more accurate historical dose
estimation (Zabel, 2006).
(C8) For childhood exposure to external radiation, the pooled ERR per Gy was 7.7 (95%
CI: 2.1, 28.7) (Ron et al., 1995). After the Chernobyl accident, most of the thyroid dose wascaused by internal exposure. In fact, reconstitution of radiation doses in a case-control study
revealed that 92.3% and 84.1% of thyroid exposures were internal radiation from 131I in
Belarus and Russia, respectively (Drozdovitch et al., 2010). In the Ukrainian arm of the firstcohort study, the ERR per Gy was 5.25 (95% CI: 1.7, 27.5) (Tronko, 2006). The ERR
estimate per Gy in the case-control study conducted in Belarus and Russia was 4.5 (95% CI:
1.2, 7.8) (Cardis et al., 2005). Thus, the ERR for external radiation exposure was compatible
with the ERR estimates for internal radiation exposure following the Chernobyl accident.
(C9) The thyroid is one of the sensitive organs to radiation regarding carcinogenesis.
Studies of children treated with external radiation demonstrated that doses as low as 0.1 Gy
could induce thyroid cancer (Shore 1992). The ERR per Gy was highest among children
exposed at the age of 0 to 4 years, and it decreased with increasing age (Ron, 1995). Among
A-bomb survivors, the risk also decreased at increasing age at exposure (Thompson, 1994).
The incidence of thyroid cancer among the population exposed after the Chernobyl accident
was inversely correlated with age at exposure in many studies (Saad, 2006). It is generally
believed that a higher risk of thyroid cancer from childhood exposure is likely due to a higher
proliferative activity of the thyroid in children. Since the proliferation rate of thyroid cells issignificantly higher before birth (Table C.1.) (Saad, 2006), it is expected that in utero
exposure may increase the incidence of thyroid cancer. However, no evidence so far has
shown a statistically significant increase, while in utero exposure exhibited a statisticallysignificant increase in the incidence rates of solid cancer (Preston, 2008). For example, Abomb survivors exposed in utero showed a similar risk for solid thyroid nodules to thatobserved in those exposed in childhood (Imaizumi, 2008). Although the information on
thyroid cancer risks associated with in utero exposure to the Chernobyl fallout is very limited,no significant increase in thyroid cancer was observed 15 years after the accident (Shibata,2001). A more-recent screening study suggested that in utero exposure may have increased
the risk of thyroid carcinoma approximately 20 years later (Hatch, 2009); however, theestimate was not statistically significant. Apparently, additional studies are required to clarify
the issue of in utero exposure (Cardis and Hatch, 2011). So far, possible explanations for thediscrepancy is that thyroid stem cell (TSC) proliferation in early fetal life may eliminate
damaged cells more efficiently, or repair DNA damage more efficiently, or enhance the ROS124

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scavenging system, and the small size of the Chernobyl in utero cohorts compared to thepostnatal Chernobyl cohorts.
Table C.1. Proliferation rate of thyroid cells (Saad 2006)
Age
Labelling index (%)
Fetal (weeks gestation)
11~15
16~20
21~25
26~30
31~35
36~40

16.3
11.7
8.1
7.6
1.9
0.4

Paediatric (years)
0~12
13~19

0.07~0.38
0.11~0.18

Adult (years)
20~39
40~60

0.08~0.11
0.08~0.09

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C.3. General features of the thyroid
C.3.1. Human thyroid development and stem cells(C10) Mice and humans possess the same set of endocrine organs, as well as the common
orthologous factors that have been implicated in the development and maintenance of the
thyroid during the embryonal period and after birth (Trueba et al., 2005). In humans, thethyroid is the largest classical endocrine gland weighing about 20~40 g in adults. The thyroid
gland is butterfly-shaped and composed of two lobes with an isthmus connecting them. The
thyroid is comprised of spherical follicles which contain colloid (Fig. C.2.). The follicles are
surrounded by a single layer of thyroid epithelial cells called follicular cells. Colloid is
produced by the follicular cells and is rich in thyroglobulin (Tg) protein. Between thespherical follicles is another type of thyroid cells, parafollicular or C cells, which secrete
calcitonin. Outside the follicles, there are two other types of cells, which are endothelial cells
and fibroblasts.
(C11) Thyroxine (T4) is the hormone synthesised from Tg by thyroid follicular cells. Afterthe sodium/iodide symporter (NIS)-dependent accumulation of iodide into the follicularlumen, iodide is rapidly oxidised by thyroperoxidase (TPO) in the presence of hydrogen
peroxide, generated by dual oxidases (Ris-Stalpers, 2006; Sumimoto, 2008), and is attachedto tyrosyl residues in Tg, followed by coupling of iodotyrosines to form T4.

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Follicular cells
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Colloid

Fig. C.2. Histological structure of thyroid follicles(C12) Human ES-cell-derived, endoderm-positive cells have been shown to express
several early thyroid markers, including thyroid stimulating hormone receptor (TSHR) andpaired box gene 8 (Pax8), but not Tg. Therefore, with the restrictions of human ES studies,
the main efforts have been directed to the identification of adult TSCs. It has been speculated
for a long time that a subpopulation of pluripotent stem cells, present in the inner cell mass ofthe pre-implantation embryo, may never differentiate; instead, these cells might persist andseed adult tissues (Weissman, 2000). Such cells can generate diverse cell types during tissue
renewal in response to environmental signals. Recently, the cells expressing markers ofpluripotency have been identified in the thyroid. The steady synthesis of octamer-bindingtranscription factor 4 (OCT4) mRNA can be detected in a culture of adult primary thyrocytes
indicative of the possible presence of cells with an embryonic-like phenotype (Thomas et al.,2006). Oct4 belongs to the family of POU-domain transcription factors abundantly expressedin embryonal tissues, whereas it is downregulated in most adult somatic cells (Pesce andScholer, 2001; Pesce, 1999). In cultures of adult thyrocytes, the expression of OCT4 mRNA
remains stable over several passages without signs of reduction, suggesting the ability of
Oct4+ cells for self-renewal and reconstitution of the population after subculturing.
Immunohistochemical and FACS analysis also confirmed the existence of Oct4+ cells, which
account for 0.2% of the total thyroid cell population. Sorted Oct4+ cells express PAX8, but
not TG mRNA, indicating that despite the synthesis of the embryonic marker of pluripotency,
the cells have already acquired some degree of differentiation (Thomas et al., 2006).
(C13) In human fetal thyroid, Pax8 is expressed from Carnegie stage (CS) 14, while Tg
appears only at CS19 (Trueba et al., 2005). It is therefore possible to speculate that
Oct4+Pax8+Tg– cells in adult thyroid may represent the group of embryonic cells, whosematuration is stalled at an embryonic age of CS14-19. These cells may be the candidates for
adult TSCs. The Oct4+ cells are not always equal and specific to stem cells because of a
mixture of stem cell-derived progenitor cells (Lan, 2007).
(C14) The presence of cells with an embryonic phenotype in the thyroid might represent asituation in which immature cells and adult thyrocytes with “facultative stem-cell properties”
coexist in the gland as indicated in the pancreas (Dor, 2004), and both may contribute totissue homeostasis. In the thyroid, the solid cell nests (SCNs) have been speculated for a longtime to be the “niche” and main repository of embryonic cells. However, only by pathologicalexamination, SCNs are considered to represent the embryonic remnants of ultimobranchialbodies (UBBs) and are composed of thyroid transcription factor 1 (TITF-1) positive C-cells

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(Cameselle-Teijeiro et al., 1994; Damante et al., 2001; Harach, 1988; Reis-Filho et al., 2003).
TITF-1 is a transcription factor, which protects the fetal primordium and UBBs against
apoptosis in the embryonal period (Damante et al., 2001; De Felice and Di Lauro, 2004; De
Felice et al., 2004; Kusakabe et al., 2006). Once activated at CS14, TITF-1 expression
remains for a lifespan in the follicular and C-cells (Ordonez, 2000; Reis-Filho et al., 2003).
Among the cells of SCNs, there is a distinct group of so-called “main cells”, which show no
TITF-1 staining. It has been suggested that the main cells of SCNs could transdifferentiate tofollicular cells under certain circumstances (Burstein et al., 2004). The presence of colloidcontaining follicles in SCNs of some patients (Harach, 1991) and in mice (Manley andCapecchi, 1995), as well as the high expression of p63 (Preto et al., 2002; Reis-Filho et al.,2003) and telomerase (Burstein et al., 2004; Preto et al., 2004) have been demonstrated in themain cells, highlighting their unique properties. p63 is usually detected in the basal andprogenitor layers of many epithelial tissues, such as breast and prostate, where it is
responsible for survival of basal layers of epithelium and stem-like properties (Di Como et al.,
2002; Preto et al., 2002; Signoretti et al., 2000; Yang et al., 1999). Also, the telomerase
elongates the protective caps at the ends of chromosomes (Hiyama et al., 1995a) and canconfer increased lifespan without eliciting a cancer-associated phenotype (Jiang et al., 1999;
Vaziri and Benchimol, 1998). It may allow the stem cells to overcome replicative senescence(Mathon and Lloyd, 2001; Wynford-Thomas, 2000) and maintain the original DNA matrix in
an intact state. Therefore, the co-expression of p63 and telomerase in the main cells of SCNs
may reflect a unique role of these cells in the thyroid and point to their stem-like properties.
This combination of features gives SCNs strong evidence for being considered as a“residence of stem cells” (Preto et al., 2004; Preto et al., 2002).
C.3.2. Tissue turnover rate(C15) The turnover rate of normal human thyroid tissue was estimated using in vitro
labelling experiments. Normal human thyroid tissues were obtained from adult patients in theage 22-56 years with solitary benign nodules or thyroid cysts. The estimated minimallabelling index (LI) was 1.34 x 10-4. Assuming an S phase of 10 hours, it corresponds to aturnover rate of 8.5 years (Coclet et al., 1989). Estimated turnover rate was between 8.5 and14.4 years, which implies that the thyroid follicular cells divide approximately 4 or 5 times
during adulthood.
(C16) Also, other studies have shown that if the culture medium is supplemented withcertain growth factors, the thyrocytes can divide up to 40 times (Curcio et al., 1994), and thatentire follicles can be regenerated through the division of a single thyrocyte and assemblingof daughter cells (Toda et al., 2003, 2001a, b). The neofolliculogenesis also occurs in
inflamed thyroid: in subacute thyroiditis, the progressive inflammation and gradualdestruction of parenchyma paralleled partial restoration through the division of differentiatedthyrocytes (Toda et al., 2000). Thus, it confirms that the proliferative capacities of thyrocytes
themselves might be enough for the turnover in normal and damaged tissue. The thyrocytes
acting as facultative stem cells are an attractive hypothesis. In this model, long-lived
thyrocytes can maintain the follicles’ structure without intermediate progeny, and they also
can accumulate multiple genetic alterations until they acquire cancer hallmarks for clonal
expansion.
(C17) The number of regenerative cells, which corresponds to either progenitors or stem
cells, was assessed by an experiment in which monodispersed rat thyroid follicular cells weretransplanted into the fat pads of thyroidectomised rats. The number of clonogens (colonyforming cells) per follicle was calculated to be approximately 3, which represents about 3%
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of all follicular cells (Clifton et al., 1978; Watanabe and Hendry, 1991). It has also recently
been shown that human thyroid follicles are able to be transplanted into severe-combined
immunodeficiency (SCID) mice (Nomura T, 2008). However, the number of clonogens inhuman thyroid has not yet been determined.
C.3.3. Age and gender specificity of tissue turnover(C18) From a temporal point of view, the human thyroid primordium starts to bud at CS12,
analogous to embryonic day 8 (E8) in the mouse, that is paralleled by the early expression of
TITF2 and PAX8 genes during the migration and differentiation of thyroid cells. Oncethyroid organogenesis is finished by 12-13 weeks gestation, the thyroid becomes functionallyactive (De Felice and Di Lauro, 2004). Follicle precursors are detectable, and Tg is secreted
into follicular spaces at this stage. Thyroid weight between 95 and 100 days gestation is 80
mg and it becomes 1,430 mg by the time of birth (Table C.2.).
Table C.2. Weight of the thyroid at fetal ages (Fisher, 1974)
Fetal age (days)
95~100105~120122~138
145~165
166~230
Term

Fetal weight (g)
75
146
203
465
1080
3270

Thyroid weight (mg)
80
131
139
261
723
1,430

(C19) The highest cell proliferation rate, as judged by Ki-67 nuclear staining, wasobserved in early fetal life (Saad, 2006). At 11 to 15 weeks gestation, Ki-67 staining was
positive in about 16% of cells. Then, it continuously decreased to 0.4% at 36 to 40 weeks
gestation. After birth, the proliferation rate was between 0.2 to 0.3% in the paediatric groupand close to 0.1% in the adult group, which are comparable to the results obtained by others(Katoh, 1995; Shimizu, 1993).
(C20) At the time of birth in the full-term newborn, an abrupt rise in the serum TSH leveloccurs within 30 minutes of delivery. This results in a dramatic stimulation of thyroid
function. Following the neonatal period, there is a gradual decrease in the T4 production rate.
In infants, it is about 5 to 6 mg/kg per day, and over the first few years, it decreases to 2 to 3
mg/kg per day at ages 3 to 9 years. This is in contrast to the rate in adults, which is about 1.5
mg/kg per day. The weight of the thyroid of the newborn is approximately 1 g and it
increases about 1 g per year until age 15 years, when it reaches the adult size of about 15 to
20 g. Average weights of thyroid glands in males in the age ranges 20-29 years and 30-69
years were 16.4 g and 18.5 g, respectively. The average weight of the female thyroid gland in
the age range 20-69 years was 14.4 g (Pankow, 1985).
(C21) As discussed in the tissue turnover rate section, in vitro labelling of human thyroid
slices demonstrated the LI of 1.34 x 10-4 for normal human follicular cells obtained fromadults between 22 to 56 years old, which corresponds to a turnover rate of the order of 8.5
years (Coclet et al., 1989). A different LI was obtained in one 13-year-old subject, 2.49 x 10-3,
which corresponds to a turnover rate of the order of 167 days. Higher LIs have also been
observed for young rats versus old rats (Sheline, 1969).
C.3.4. Mouse thyroid development and embryonal stem cells
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(C22) The mouse is the best-studied animal model for embryonic development of the
thyroid gland. During gastrulation, the epiblast, consisting of multipotential cells, generates
three embryonal layers: endoderm, mesoderm and ectoderm. Cells within the specified areasof the endoderm respond to inductive signals and differentiate toward a specific lineage, suchas pancreatic or hepatic (Fukuda and Kikuchi, 2005; Tam et al., 2003). The formation of the
thyroid begins at embryonic day 8, when the thyroid primordium from a ventral outpocketingof the pharynx starts to migrate caudally to join with lateral outpocketings from the fourthpharyngeal pouch, called UBBs. In the mouse, these series of events take place before E14(De Felice and Di Lauro, 2004; De Felice et al., 1998; De Felice et al., 2004). The functional
differentiation of the thyroid gland starts at E14, thus completing organogenesis. From thismoment, thyroid-specific genes such as TPO, Tg and TSHR become transcriptionally active,
followed by NIS expression and follicle formation at E16 (Postiglione et al., 2002).
(C23) Considering that thyroid parenchyma is composed of the follicular and C-cells,
differentiation of thyrocytes induced in the culture may trigger the differentiation of C-cells
as well. NeuroD1 is the neuroectodermal transcriptional factor, expressed in C-cells from E15
and known to coordinate the terminal differentiation of neuroendocrine cells by controllingthe cell cycle and activating the transcription of hormonal genes (Kameda et al., 2007; Mutoh
et al., 1997). In the culture of ES cells, the thyroid differentiation is not associated with theincrease of NeuroD1, showing that thyrocytes and C-cells are developmentally dissociatedand require distinct factors to enter the differentiation programme, at least under theseexperimental conditions (Lin et al., 2003).
C.3.5. Cellular features(C24) Thyroid specific gene expression has been well-studied in the mouse. In vivo, Titf2
and Pax8 are involved in the early stages of thyroid formation. During mouse development,
Titf2 is expressed within E8-13 and is responsible for functional repression of thyroidspecific genes until migration of the thyrocytes through the mesoderm has been fullycompleted. The Titf2 knockout mouse develops normal follicular and C-cells, whereas
thyroid primordium fails to migrate and remains ectopically attached to the pharynx (DeFelice et al., 1998; Zannini et al., 1997). PAX8 is crucial for fetal thyrocytes from E10. In
the PAX8–/– mouse, the thyroid is the only organ which cannot develop, while other tissues
easily overcome the effects of the gene knockout, indicating its importance for survival offollicular cells (Mansouri et al., 1998; Plachov et al., 1990). In the murine fetal thyroid, both
PAX8 and Titf2 are simultaneously expressed only for a short time during E10-13, before thetranscriptional activation of NIS, TPO and TSHR (De Felice and Di Lauro, 2004; Japon et al.,1994). In culture, the concerted expression of PAX8, Titf2 and TSHR in embryoid bodies
clearly indicates cell differentiation into the thyroid lineage; however, the temporal gene
expression in this system is different from the temporal pattern in vivo.
(C25) The TSH/TSHR axis regulates the expression of thyroid-specific genes, such as TPO
and NIS (Kogai et al., 1997; Levy et al., 1997; Zarrilli et al., 1990), whereas it is not required
for the onset of TG expression in mouse fetal thyroid (Postiglione et al., 2002). From thisviewpoint, the acquisition of thyro-phenotype by cells of embryoid bodies would probably
occur under the control of TSH. The addition of TSH to the growing ES cells not only
augments NIS and TPO expression, but also induces the expression of TSHR and PAX8, thus
confirming the leading role of TSH in maintaining the thyroid-like phenotype. In this model,
and likewise in vivo, TSH shows no effect on induction of TG mRNA synthesis in embryoid
bodies (Lin et al., 2003). Therefore, despite differences in the temporal pattern of gene
129

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5421	
  
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5436	
  
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expression in an in vitro model, the main mechanisms involved in thyroid differentiation
remain highly similar to those occurring in vivo.
(C26) The directed differentiation of ES cells toward the thyroid lineage enables us to lookat early thyroid progeny and to trace the fate of fetal follicular cells under different conditions.
Current approaches in manipulation with mouse embryos allow isolation of ES cells fromblastocysts and maintenance of them in an undifferentiated state. When plated on feeder
layers of mitotically inactivated fibroblasts, the cells keep their embryonic morphology andabundantly express the stem cell marker Oct4 (Pan et al., 2002; Sun et al., 2006; Tai et al.,2005). However, when appropriately prompted to differentiate, ES cells undergo dramaticchanges in gene expression profile and can give rise to the thyrocyte-like cells (Lin et al.,2003). This is seen as a formation of embryoid bodies in the culture plate and the expressionof PAX8, NIS and TSHR mRNA at day 6 of differentiation, and the appearance of Titf2,
Pax8 and Tshr proteins at day 8. The thyrocyte-like cells protrude from the main cell mass ofthe embryoid body and tend to form a monolayer of differentiated cells (Lin et al., 2003). In a
study to facilitate the tracing of early thyroid cells, ES cells have been genetically modified
and the GFP-NEO construct has been introduced to ES cells under the control of a TSHR
promoter, therefore allowing visualization and isolation of the early thyroid progeny by cellsorting (Arufe et al., 2006). In genetically modified ES cells, the acquisition of thyroid
phenotype by ES cells leads to appearance of GFP-positive cells at day 2 of differentiation.
When sorted, GFP fluorescent cells show characteristic features of the thyroid phenotype:they express NIS, TPO and assemble into follicle-like structures. While TSH-directed thyroid
cell differentiation from ES cells has been well documented, ES cells exposed to activin A
also underwent endoderm differentiation, which is a prerequisite for thyroid differentiation
with the aid of TSH and IGF1 (Ma, 2009).
(C27) More recently, recombinant ES cell lines, in which TITF-1 and Pax8 expressions are
independently regulated by the addition of doxycyclin, were established (Antonica, 2012).
Transient concurrent expression of TITF-1 and Pax-8 efficiently promoted the expression of
TSHR, TPO, NIS and TG mRNA. In combination with TSH, ES cell committed thyroid
follicular cells gave rise to 3D structures reminiscent of thyroid follicles, which are functionalin vivo as demonstrated by transplantation into hypothyroid mice.
(C28) The ability to generate and isolate embryonal thyrocytes is a strong impulse forfurther study of the signals and mechanisms involved in maturation of TSCs. The molecularmechanism of mouse thyroid carcinogenesis should be examined using more sophisticatedapproaches, especially focussing on age-dependent cell characterisation.

5441	
  

C.4. Radiosensitivity

5442	
  
5443	
  
5444	
  
5445	
  
5446	
  
5447	
  
5448	
  
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5450	
  
5451	
  
5452	
  

(C29) The thyroid is very radioresistant regarding dysfunction. Several studies
demonstrated that thyroid dysfunction, such as hypothyroidism and thyroiditis, were caused
only by high-dose radiation exposure from radioactive iodine therapy and external
radiotherapy. For example, acute radiation thyroiditis occurs within 2 weeks after exposure of
the thyroid to radioiodines. The symptoms include inflammation and necrosis of some or allcells in the thyroid gland. From several clinical observations, clinically significant acuteradiation thyroiditis may not be seen at thyroid doses below approximately 200 Gy from 131I
(Maxon and Saenger, 2000). Above this threshold dose, each 100 Gy increment is estimatedto develop acute radiation thyroiditis in an additional 5% of persons. Hypothyroidism is acondition caused by insufficient thyroid hormone from the thyroid gland. A linear doseresponse relationship was observed between radiation doses to the thyroid from 131I and the130DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
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5479	
  
5480	
  
5481	
  

probability of development of hypothyroidism (Maxon, 1977). The lowest dose fordeveloping hypothyroidism was approximately 270 Gy from 131I (Maxon and Saenger, 2000).
(C30) Dormant thyroid cells are very radioresistant, but their radiosensitivity to a
proliferative stimulus in vivo was determined by transplantation of thyroid cells into the fatpads of thyroidectomised rats. The dose-response curve for the clonogens gave a mean D0
value of 197 cGy (Mulcahy et al., 1980). This is lower but of the same order as the value of
350 cGy measured by assessing the survival of whole transplanted thyroid follicles(Watanabe and Hendry, 1991). Although the number of clonogens in human thyroid is still tobe determined, the thyroid epithelial cells derived from surgically removed thyroid tissues
have been cultured in vitro, and radiation sensitivity determined by colony forming assay.
The mean D0 dose was estimated to be 0.93-0.94 Gy (Hiraoka et al., 1985; Miller et al., 1987).

5482	
  

C.5. Mutagenesis

5483	
  
5484	
  
5485	
  
5486	
  
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(C33) Oncogenic mutations found in thyroid cancer are different depending upon thepatients' age at exposure and diagnosis, clinicopathological manifestations, and theindividual's genetic characteristics. According to the current histological classification,
papillary, follicular, medullary, and undifferentiated carcinomas are the major diagnostic
types of malignant thyroid tumours. Among them, PTC is the most prevailing type of thyroid
cancer in both children and adults (Table C.3.) (Ciampi and Nikiforov, 2007; Fagin and
Mitsiades, 2008; Yamashita, 2007).
(C34) Abnormal thyroid follicles with reversed polarity have been induced by radiation(Watanabe and Hendry, 1994). These were produced by irradiating rat thyroids in vivo to 5.5
Gy, disaggregating the follicles, injecting the cells into fat pads of thyroidectomised
recipients, and scoring the frequency of abnormal follicular structures at 6 weeks later. These
dysplastic follicular structures imply the induction of stable mutants, reminiscent of theradiation-induced ductal dysplasias produced in a similar mammary tissue model (Annex B)
and dysplastic crypts induced in the colon (Annex D).

C.4.1. Characteristics of damage response at the cell level(C31) DNA damage checkpoint activation was examined in primary thyroid cells obtained
from thyroid tissues, which were taken by thyroidectomy from patients with hyperthyroid
diseases. A p53-p21-dependent G1 checkpoint activation was demonstrated in response toradiation exposure in vitro (Namba, 1995). The ATM-dependent DNA-damage checkpoint
pathway was active, because phosphorylation of histone H2AX, which is one of thedownstream effectors of the ATM pathway, was demonstrated in primary human thyroid
cells (Galleani et al., 2009). The γH2AX assay revealed that DNA repair kinetics in primarythyroid cells was comparable to those reported in other normal human cells.
(C32) There is, however, little information available for human TSCs. Recently, it wasdemonstrated that human thyroid tissues could be successfully maintained in the improved
SCID mice up to 2 years (Nomura, 2008). Histological features of human thyroids in
improved SCID mice were well preserved. After irradiation, follicles disappeared and thyroid
hormone secretion was dramatically decreased in a dose-dependent manner. 3D-reconstituted
follicles were demonstrated from thyroid cells embedded in collagen (Toda et al., 2001a).
These approaches could be applicable for mimicking thyroid folliculogenesis in vivo.

Table C.3. Histological types of human thyroid cancer (Yamashita and Saenko, 2007).
Tumour types
Prevalence (%)
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Children and adolescents

Adults67~98
4~23
2~8; 17
<0.1

85~90
<10
3
2-7

Papillary carcinoma
Follicular carcinoma
Medullary carcinoma
Poorly differentiated andundifferentiated carcinoma
5499	
  
5500	
  

5501	
  
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5520	
  
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5523	
  
5524	
  

Table C.4. Prevalence of genetic alterations in human thyroid cancer (Yamashita, 2007).
Prevalence (%)
Molecular alterations
Children and adolescents
AdultsGene rearrangements:
RET/PTC
NTRK1
AKAP9-BRAF
PAX8-PPARG

38~87
5~11
11
Unknown

0~35
5~13
1
0~50

Point mutations:
BRAF
RAS family
GNAS
p53

0~16
0~6
0
0~23

25~69
0~43
~11
0~20

(C35) Gene rearrangements are the most prevailing genetic alterations in childhood PTC(Fagin, 2004; Fenton et al., 2000; Nikiforov, 1997) (Table C.4.). Fusions of the RET protooncogene with several partner genes, which collectively been designated RET/PTC (Fusco etal., 1987; Grieco et al., 1990), are the most frequent gene rearrangement in PTC in children.
The RET gene encodes protein for a membrane receptor tyrosine kinase (RTK). It consists ofthree functional domains: an extracellular ligand-binding domain, a transmembrane domain,
and a tyrosine kinase domain. Binding of the ligands, growth factors belonging to the glial
cell line-derived neutrophic factor family, causes receptor dimerisation, autophosphorylationof the intracellular domain, and activation of tyrosine kinase activity. The partner geneproducts are highly expressed in thyroid follicular cells, and they possess coiled-coil domainsthat enable homodimerisation of RET/PTC proteins. As a result, RET/PTC proteins areactivated independently of the ligand. Other types of rearrangements include juxtaposition of
the A kinase anchor protein 9 gene (AKAP9) and v-raf murine sarcoma viral oncogene
homologue B1 (BRAF), designated AKAP9-BRAF, and rearrangement of the neurotrophic
tyrosine kinase receptor type 1 (NTRK1) gene (Table C.4.).
(C36) An age-dependent disproportion in the prevalence of gene rearrangements and point
mutations is demonstrated in childhood and adult PTC (Yamashita, 2007). In contrast to
RET/PTC rearrangement, point mutations are rather infrequent events in childhood thyroid
carcinoma irrespective of radiation exposure (Table C.5.). For example, a point mutation incodon 600 of the BRAF gene is the most prevailing mutation in adult PTC (Xing, 2005).
However, it is quite uncommon in paediatric patients (Kumagai et al., 2004; Lima, 2004;
Nikiforova, 2004).

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Table C.5. Prevalence of BRAF mutation and RET/PTC rearrangements in PTC (Xing, 2005).
Number of cases/Total (%)
BRAF mutation
RET/PTC rearrangements
Radiation-exposed

Non-exposed

Radiation-exposed

Non-exposed

7/109 (6)2/55 (4)92/175 (53)29/56 (52)

(C37) After the Chernobyl accident, the highest risk for radiation-induced thyroid cancer
was observed among children exposed at the age of 0-4 years. The childhood PTC showed asignificantly higher prevalence of gene rearrangement than later-onset tumours. In particular,
RET/PTC3 and less frequently RET/PTC1 rearrangements were predominant in PTC in
paediatric patients (Fugazzola et al., 1995; Klugbauer, 1995; Nikiforov, 1997; Williams,
2009). Direct connection between radiation exposure and induction of thyroid tumours
suggests that radiation-induced mutagenesis is likely involved in tumour development. In Abomb survivors, RET/PTC rearrangements have been correlated with the radiation dose
(Hamatani et al., 2008); however, a similar correlation was not found in Russia after the
Chernobyl accident (Tuttle, 2008). In vitro experiments using primary thyroid tissuetransplanted into SCID mice demonstrated radiation-induced RET/PTC rearrangements (Itoand Cotsarelis, 2008; Mizuno, 2000). Chromosomal loci participating in RET/PTC1
rearrangement have been shown to be in spatial proximity in human interphase thyrocytes,
which possibly facilitates the rearrangements by radiation exposure (Nikiforova, 2000). Thus,
while radiation exposure is a potent inducer for gene rearrangements, the RET/PTC
rearrangements observed in childhood thyroid cancers after the Chernobyl accident showed
less evidence of a common radiation signature. Therefore, they may not be a directconsequence of radiation exposure, rather it augments the frequency of the same eventsoccurring in sporadic cases.
(C38) In relation to mutagenesis, another unique characteristic of the thyroid gland is theevidence that thyrocytes continuously generate ROS, particularly hydrogen peroxide (H2O2),
in addition to those ROS produced by mitochondrial aerobic respiration (Krohn et al., 2007).
Hydrogen peroxide is a ROS that is a key component of hormonogenesis, and it is generated
by dual oxidases (DUOX1/2), also known as thyroid oxidase (ThOX1/2) (Sumimoto, 2008).
High concentrations of H2O2 together with oxidised iodine, which is converted from iodide
via a reaction catalysed by TPO, are required for iodination of tyrosine residues on Tg. A
variety of ROS defense systems are upregulated in thyrocytes, although there are studies
showing that excess H2O2 levels could be hazardous for the genome of thyroid cells (Poncin,2009). For example, an antibody against 8-oxo-2'-deoxyguanosine (8-OHdG), one of thewell-known DNA adducts, showed the most intensive staining in the follicular cells aroundthe lumen, where H2O2 is generated (Maier, 2006). Such oxidative stress is likely to be
involved in spontaneous DNA damage induction, which possibly results in the elevation of
the spontaneous mutation rate demonstrated in the thyroid gland from LacZ transgenic mice
(Maier, 2006). Thus, although there is no study showing a direct connection between elevated
ROS level and BRAF mutations so far, oxidative stress during hormone production could bea cause of mutagenesis in thyroid cells (Krohn et al., 2007).
(C39) While several studies have described RET/PTC rearrangements in childhood thyroid
cancer after the Chernobyl accident, the principal difference in terms of molecular events
between sporadic and radiation-induced thyroid cancers needs to be determined in futurestudies. The molecular analysis of the genomic sequence of RET/PTC fusion point foundevidence for the use of NHEJ to repair radiation-induced DSBs (Gandhi et al., 2010).
However, this would not be sufficient enough to identify the role of radiation in RET/PTC
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rearrangements. Furthermore, the most common point mutation in the BRAF gene, although it
is not the major event in childhood thyroid cancer after the Chernobyl accident, involves T:A
to A:T transversion, which could not be the transversion induced by oxidative DNA damage,such as 8-oxo-guanine (Loon et al., 2010; Nakabeppu, 2006). Thus, at the present time, it isimprobable to identify radiation signatures in RET/PTC rearrangements and in BRAF
mutation. Since the mutation type corresponds more to age-of-onset of thyroid cancer rather
than radiation, the role of radiation for the induction of oncogenic mutation remains to beelucidated.

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C.6. Summary

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(C40) Fundamental knowledge of thyroid stem cells: The term “stem cell” is widely used todescribe a cell capable of both prolonged self-renewal and differentiating into one or morefunctional cell types. Their property of symmetrical cell division is a critical phenomenon. So
far, there is no definite marker to identify thyroid-specific stem cells from embryonic as wellas adult thyroid glands. The thyroid gland, despite being an epithelial organ, is believed toshow an extremely low rate of self-renewal in vivo, and primary thyrocytes barely proliferate
in vitro. The neonates and children, however, have a growth capacity for every organ in thebody, including the thyroid gland. Preliminary data also suggest that primary cultured human
thyroid cells contain “proliferative follicular cells” in certain culture conditions (Suzuki,2011). Furthermore, goitorous thyroid glands, either simple or multinodular, or benign ormalignant, may have relatively a large number of progenitor or stem-cell-like thyroid cells.
This evidence suggests a different capacity and reaction between neonate/childhood and adultthyroid cells.
(C41) Recent review articles provide some hints and insights into the pure isolation of
TSCs (Lin, 2007; Thomas, 2008). However, the niche or microenvironment of TSCs,
including the time and spatial effects of paracrine factors, are unknown or ignored in TSC
research in vitro. Specific culture conditions with EGF and bFGF are suitable for obtaining
thyroid spheroids (Lan et al., 2007), which are similar in morphological appearance and longterm culture characteristics to neurospheres (Fierabracci et al., 2008). These thyrosphere celllines may be a good tool for further TSC research. Their purification and analysis ofinteractions between signalling pathways probably would be the main future direction in TSC
research.
(C42) Questions from human thyroid carcinogenesis by radiation: In view of a rapid
increase of childhood thyroid cancers after the Chernobyl accident (Demidchik et al., 2007),
identification of the effect of radiation on TSCs is being sought (Yamashita, 2007). Several
reports implied that accidental or medical radiation exposures are likely cause the RET generearrangements (Bounacer et al., 1997; Fugazzola et al., 1995; Klugbauer, 1995). In vitro
studies have confirmed that radiation is able to induce RET/PTC rearrangements (Ito et al.,1993b; Mizuno, 2000). However, such RET/PTC rearrangements are observed not only inthyroid cancer from radiation exposed patients, but also those occurring in unexposed
thyroids (Yamashita et al., 2007). The principal difference in terms of molecular events
between sporadic and radiation-induced thyroid cancers is not yet clear. Therefore, it is stillquestionable whether exposures to relevant doses of ionising radiation are the primary cause
for the RET/PTC rearrangements. Since the mutation type corresponds more to age-of-onsetof thyroid cancer rather than radiation, the role of radiation for the induction of oncogenic
mutation remains to be elucidated. In this regard, a critical question is whether or not the lateoccurring thyroid cancers of the childhood-exposed population in the Chernobyl area carry
BRAF mutations.

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(C43) The characteristics of the thyroid gland and its radiation response described in thisAnnex are summarised in Table C.6.
Table C.6. Characteristics of the thyroid gland
Stem cells and other cells

The human thyroid primordium starts to bud at Carnegie
stage (CS) 12, and Pax8, a marker for early thyroid, isexpressed from CS14, indicating that thyroid-committed
stem cells already exists by CS12. In adults, in vitro
labelling of human thyroid slices demonstrated the LI of
1.34 x 10-4, which corresponds to 0.2% of Oct-4 positive
cells in thyroid tissue. No such information is available fornewborn, infant, adolescent, and other aged periods.

Turnover rate

From in vitro labelling experiments, and assuming an S
phase of 10 hours, a tissue turnover rate is estimated to be
between 8.5 and 14.4 years. The same labelling experiments
demonstrated that a subject from a 13 year-old patient
showed a turnover rate of the order of 167 days.

Tissue architecture

Thyroid organogenesis is finished by 12-13 weeks gestation,and the thyroid becomes functionally active. Follicle
precursors are detectable, and Tg is secreted into follicularspaces at this stage. The solid cell nest could be a niche for
TSCs.

Cellular radiosensitivity

D0 values of 0.9-3.5 Gy have been reported for thyroid
clonogenic cells/regenerative cells. No reports havecompared the radiosensitivity of stem and progenitor cells.

Susceptibility to radiation The ERR of thyroid cancer is 1.28 per Gy at age 60 years
carcinogenesis
after exposure at age 10 years. An age-dependent decreasein the ERR is clear. There is little cancer risk for those
exposed after the age of 20.
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ANNEX D: DIGESTIVE TRACT STEM CELLS

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D.1. Radiation induced cancer in the digestive tract

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(D1) Evidence that ionising radiation causes cancer of the GI tract in humans has beenreviewed (Boice and Fry, 1995; UNSCEAR, 2000, 2008). The evidence comes fromepidemiological studies of the Japanese A-bomb survivors (Ron et al., 1994; Thompson et al.,1994), patients given radiotherapy for cervical cancer (Boice et al., 1987; Boice et al., 1985;
Boice et al., 1988), and patients treated with radiation for benign conditions such asankylosing spondylitis (Darby et al., 1987), gynaecological disorders (Darby et al., 1994;
Inskip et al., 1990), and peptic ulcer (Griem et al., 1994). The studies of A-bomb survivors
have followed subjects prospectively for over 40 years at the level of mortality (Davis et al.,
1989; Shimizu et al., 1990) as well as cancer incidence (Thompson et al., 1994; Ozasa et al.,2012). The average whole-body dose was relatively low, approximately 0.23 Gy, but the dose
distribution ranged up to 4 Gy, permitting dose–response evaluations. This is the single mostinformative study on radiation risks to date, including exposures of both men and women,
and children and adults. Its major limitation is that risks apply to acute brief radiation
exposures, and is not directly applicable to the chronic or periodic exposures experienced indaily life from environmental, occupational, or medical sources.
(D2) The risk of radiation-induced cancer in the mouth and small intestine is vastly
smaller than in the other regions of the alimentary tract, and they are not given specific valuesof tissue weighting factor but are included in remainder tissues (ICRP, 2007a). This sectionof the report will focus on oesophagus, stomach and colon/rectum.
(D3) The study of patients irradiated for ankylosing spondylitis was the only study of
medically exposed populations to report a significant risk of radiation-associated oesophageal
cancer. These patients received the highest estimated dose of 4 Gy, and were at asignificantly increased risk of mortality in comparison with the general population. The RR at
1 Gy was estimated as 1.3 (Darby et al., 1987) and AR was estimated as 0.21 x 10-4 PY/Gy.
There was some association of radiation exposure and oesophageal cancer in the LSS. Since
oesophageal cancer is relatively infrequent, there was insufficient statistical sensitivity todetect an excess in the several low-dose occupational studies. There are insufficient data tocharacterise the shape of the dose-response curve (UNSCEAR, 2008). Information on the
distribution of cancers arising in the oesophagus suggests that the greatest proportions areseen in the distal region.
(D4) Stomach cancer accounted for 16% of the total excess cancer attributable toradiation in A-bomb survivors (Thompson et al., 1994). The ERR of fatal stomach cancer at 1
Gy was estimated from the A-bomb studies as 0.65 in females and 0.2 in males, with EAR of
3.3 in females and 2.1 x 10-4 PY/Gy (Preston et al., 2003b). The ERR per Gy declined
substantially with increasing age at exposure, but declined very little with increasing attained
age. Significant excess occurred in two other series: cervical cancer patients (Boice et al.,1988) and peptic ulcer patients (Carr et al., 2002). In the US peptic ulcer study, the ERR per
Gy was 0.20 (95% CI: 0, 0.73) based on 11 stomach cancer deaths in 309 patients irradiatedwith doses of ≤10 Gy.
(D5) In general, with whole body exposures, large regional differences in dose between
different parts of the stomach would not be expected, and in that scenario, it is reasonable tocalculate an average dose. The target was taken to be the stem cell zone, treated as acontinuous uniform layer in the stomach wall (ICRP, 2007).

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(D6) The small intestine is very resistant to cancer induction by radiation. There are a few
individual medical studies but there are no reliable estimates of ERR (UNSCEAR, 2008).
(D7) Colon cancer has occurred in excess in most irradiated populations, with the notable
exception of cervical cancer patients (Thompson et al., 1994). Conceivably, at the cytotoxic
doses used to treat cancer of the uterine cervix, cell killing prevented significant celltransformation (Boice et al., 1987). Estimates of RR at 1 Gy ranged from 1.13 to 1.67, and
the AR ranged from 0.45 x 10-4 PY/Gy to 2.18 x 10-4 PY/Gy.
(D8) Colon cancer incidence was calculated for 1950–1980 in the A-bomb LSS tumour
cancer registry. Very similar linear dose-response coefficients were obtained for cancers
located in the caecum and ascending colon (ERR per Sv = 0.80, 90% CI: 0.07, 1.96),
transverse and descending colon (ERR per Sv = 1.09, 90% CI: 0.17, 2.59), and sigmoid colon(ERR per Sv = 0.96, 90% CI: 0.33, 1.87) (Nakatsuka et al., 1992). There was a significant
decrease in ERR per Sv by age at exposure. In the latest follow-up of the LSS cohort forcolon cancer mortality during the period 1950–1997, the estimate for ERR per Gy based on alinear model, for exposure at age 30 with no assumed variation by attained age, was 0.54
(90% CI: 0.13, 1.2) for males and 0.49 (90% CI: 0.11, 1.1) for females, with a 25% decrease
per decade of age at exposure (Preston et al., 2003c). UNSCEAR (2000) concluded that there
was strong evidence of an ionising radiation effect on colon cancer risks that was consistent
with a linear dose response, and this was reconfirmed more recently (UNSCEAR, 2008).
(D9) In the analysis of cancer mortality among A-bomb survivors (Preston et al., 2003b),
rectal cancer mortality was not associated with radiation dose among men. However it was
positively and significantly associated with dose among women (ERR per Gy = 0.75, 90%
CI: 0.16, 1.6), for exposure at age 30 years. Increased rectal cancer incidence has also been
observed among cervix (Kleinerman et al., 1995) and prostate cancer patients (Baxter et al.,2005) given radiotherapy. UNSCEAR (2008) concluded that it was difficult to characterise
radiation-related risk of rectal cancer at doses under about 1 Gy, but it seemed reasonably
clear that there was a radiation-related excess risk for rectal doses of tens of Gy.
D.1.1. Data for internal exposure(D10) Very limited information is available on cancer induction in the alimentary tract byingested radionuclides. Tumours induced by internal irradiation have been observed in theintestinal tract of rodents after administration of 137Cs, 95Nb, 144Ce, 90Y, and 106Ru (Casarett,1973). Intestinal polyps in dogs and rats fed 210Po or 144Ce were also reported, mostly
occurring in the large intestine, with a tendency to malignant change and showing different
latencies in the two species (Lebedeva, 1973). UNSCEAR (2000) reviewed the sparseevidence from human follow-up studies.
(D11) For the purposes of the ICRP report on the human alimentary tract (ICRP, 2007),
ingested radionuclides were assumed to deliver dose uniformly during their transit throughthe oesophagus. Dose was averaged for the oesophagus assuming a uniform distribution oftarget cells throughout the basal layer of the stratified squamous epithelium.
(D12) UNSCEAR (2000) reviewed data for stomach cancer in patients treated with 131I for
hyperthyroidism. A significant excess in terms of incidence and mortality was reported in a
Swedish study, with risks consistent with the estimate from the A-bomb study. However,UNSCEAR (2000) cautioned that because of the small numbers of stomach cancers anduncertainties in the risk estimate for 131I exposure, it was not possible to draw conclusions
about the relative effect of acute external and protracted internal radiation exposures.
(D13) Stomach cancer was shown to be significantly increased in radon-exposed cohorts ofunderground miners (Lo et al., 1995). However, there was no trend in stomach cancer
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mortality with the low levels of cumulative radon exposure to the stomach, and excesses ofstomach cancer have been reported for other groups of miners. This suggests that factors
other than radon exposure were responsible in each case. Female radium dial painters starting
work after 1930 showed an increase in stomach cancer mortality (Stebbings et al., 1984), but
those starting work before 1930, with generally higher radium exposure, did not show anincreased risk. These data do not provide convincing evidence of stomach cancer inductionby α-emitting radionuclides.
(D14) Concerning internal exposures to low-LET (131I) and high-LET radiation (radon andradium), UNSCEAR (2000) noted that the low doses to the colon did not allow conclusions
to be drawn

5728	
  

D.2. General features of the digestive tract

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(D15) The length of the oesophagus, from the pharynx to the stomach, is typically in therange of 23–30 cm in adult males and 20–26 cm in adult females (ICRP, 2002). Autopsy
measurements on infants and children indicate an oesophageal length of approximately 8–10
cm at birth, 12 cm at 1 year, 18 cm at 10 years, and 19 cm at 15 years (ICRP, 1975). The
similarity in the values for 10- and 15-year-old children is inconsistent with the rate of
growth of the upper body during that period, and suggests that the subjects may not havebeen representative.
(D16) The length of the oesophagus has been determined in a number of modern studies byexternal imaging techniques. Reported values vary considerably, apparently due mainly todifferences in the definition of ‘oesophageal length’ in medical studies and, to a lesser extent,
to intersubject variability. In a study of 51 normal adults (27 males and 24 females) from theUS, oesophageal length, defined as the average distance from the proximal end of the upper
oesophageal sphincter and the distal end of the lower oesophageal sphincter, was given as
28.3 ± 2.4 cm (Awad et al., 1999). On the basis of these data, values for the length of theoesophagus were given in Publication 89 (ICRP, 2002) as shown in Table D.1.
Table D.1. Reference values for length of the oesophagus (cm)
1 year 5 years 10 years
15 years
adult
male
femalemale
female
10
13
18
23
27
26
28
26
ICRP Publication 89, 2002.
Newborn

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(D17) The oesophageal wall of the adult is approximately 3.5–5.6 mm thick (ICRP, 1975).
The stratified squamous epithelium lining the oesophagus, illustrated in Fig. D.1, is taken to
be 200 µm thick in the adult. The target layer is assumed to be at a depth of 190–200 µm. The
oesophageal wall of the newborn is thinner than that of the adult, but the epithelium thickens
rapidly after birth, and the depth of the target layer is assumed to be independent of age forthe purpose of this report. The presence of a layer of mucus on the lumenal surface of theoesophagus has been ignored. The thickness of the mucus layer, of perhaps 10–30 µm, iswithin the range of uncertainties in the overall average depth of the basal cell target layer.
The epithelial lining of the oesophagus is a thick layer of protective tissue, many cells deep,classed as non-keratinised stratified squamous epithelium (Fig. D.1.). The basal layer
contains stem cells and proliferative cells. The diameters of the oesophagus were given in
Publication 100 (ICRP, 2007) as shown in Table D.2.
Table D.2. Assumed values for the internal diameter of the oesophagus (cm) (ICRP, 2007).
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Newborn

1 year

5 years

10 years

15 years

Adult

0.5

0.6

0.7

0.8

1

1

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Fig. D.1. Illustration of the cross-sectional structure and dimensions of the human oesophageal
stratified squamous epithelium (ICRP, 2007). Courtesy of Chris Potten, Epistem Ltd, UK.

(D18) The stomach is highly variable in volume, being capable of distension toaccommodate a large amount of ingested material. It is connected to the oesophagus at the
cardiac sphincter and to the small intestine at the pyloric sphincter. The stomach wall exhibits
numerous longitudinal folds, or rugae, when the organ is not distended with food. For the
purpose of this report, it was assumed that the stomach is a simple sphere of volume 175 cm3in adults. Volumes at different ages (Table D.3.) were based on values of mucosal surface
area given in Publication 23 (ICRP, 1975). To calculate age-dependent volumes, a constantrelationship was assumed between measured mucosal areas and the surface area of thereference spherical stomach (volume 175 cm3).
(D19) The gastric epithelium is a single layer of cells, continuous with the basal layer ofthe stratified epithelium of the oesophagus. The lining of the stomach is indented bynumerous pits that supply several million tubular glands. The glands are divided into three
categories: the cardiac glands occur in the first 5–40 mm from the cardiac orifice; the pyloric
glands occur near the intestine; and the gastric glands lie between these two regions. The cellsof the cardiac and pyloric glands all appear to be of the mucous type. The epithelium of thegastric glands is more diversified, containing enzyme- and acid-secreting cells as well as
mucous cells.
(D20) The gastric epithelium is a single layer of cells, a simple or unilaminar epithelium,
which lines numerous glandular indentations into the stomach wall (the gastric pits) (Fig.
D.2.). Differentiated epithelial cells within the gastric pits secrete hydrochloric acid (oxyntic
cells), digestive enzymes (zymogen cells), and mucous. The stem cells are assumed to be
towards the neck of the gastric pit at approximately one-third of the total pit depth (Fig. D.2).

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Fig. D.2. Illustration of the cross-sectional structure and dimensions of a typical gastric gland in thehuman stomach, lined with a single layer of columnar epithelial cells (ICRP, 2007). Courtesy of Chris
Potten, Epistem Ltd, UK. (permission needed)

Table D.3. Values for the volume of the stomach (cm3)a. Taken from ICRP Publication 100(2006).
Newborn
1 year
5 years
10 years
15 years
Adult
30

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40

60

80

120

175

a

Calculated using rounded values for mucosal surface area based on data given in ICRP Publication
23 (ICRP, 1975): 150 cm2 at 3 months (applied here to newborn), 200 cm2 at 1 year, 250 cm2 at 5
years, 300 cm2 at 10 years, 400 cm2 at 15 years, and 525 cm2 in adults.

(D21) The mucosal lining of the stomach is of variable thickness. However, for dosimetricpurposes, in situations of retention of radionuclides in the stomach wall, the source region istaken to extend to a depth of 300 µm into the mucosa. Fig. D.2. illustrates the cross-section ofa typical gastric gland, showing that the proliferative cells, and target stem cells, are thought
to be towards the upper lumenal ends of the glands. A target depth of 60–100 µm is assumed
to apply at all ages as a uniform target at this depth over the entire inner surface of thestomach.
(D22) Data on the large intestine, particularly data related to the motility of the lumenal
contents, are often reported in terms of the right colon, left colon, and rectosigmoid. The rightcolon is defined as the ascending colon, including the caecum, plus the proximal half of thetransverse colon. The left colon is defined as the distal half of the transverse colon plus thedescending colon. The rectosigmoid is defined as the sigmoid colon plus the rectum.

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(D23) Central estimates for the physiological length of the large intestine of newborns andadults are approximately 45 cm (range 20–70 cm) and 110 cm (range 91–125 cm),
respectively (ICRP, 1975). These central estimates are used in ICRP Publication 89 (2002) as
reference values for newborn infants and adults. Values for children aged 1–15 years are
based on the assumption that the length of the large intestine is linearly related to body height(see Table D.4.).
(D24) The diameter of the large intestine varies along its length, reducing from caecum torectosigmoid. The values used in this report are given in Table D.5., based on data reviewed
in ICRP Publication 23 (1975). As for the small intestine, it is assumed that the internal
diameters of the regions in infants are one-half of the values used for adults, and intermediate
values are used for 1–10-year-old children.

Fig. D.3. Illustration of the cross-sectional structure of the epithelial lining of the human large
intestine, showing crypt and stem cell position (ICRP, 2007). Courtesy of Chris Potten, Epistem Ltd,UK.)

(D25) In the large intestine, deep, straight crypts penetrate into the lamina propria from alumenal intercryptal plate; there are no villi. The stem cells are in the base of the crypts; there
are no Paneth cells as found in the small intestine. Cell division leads to a constant flow ofcells up the crypts, with loss of cells in the large intestine from the intercryptal plate. It has
been reported that there are about 82 cells per crypt column, about 46 cells per crypt
circumference, about 2,250 cells per crypt, with >5-9 transit cell generations (Potten, 1995).
(D26) Potten (see ICRP 100, 2006) measured crypt depths of 311 ± 9 µm for the ascending
colon (48 crypts from one adult), 358 ± 16 µm for the sigmoid colon (28 crypts from three
adults), and 245 ± 10 µm for the rectum. It was assumed here that the target layer is at a
depth of 280–300 µm at all ages. As for the small intestine, the target cells were taken to141

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form a continuous layer at this depth in a cylindrical tube (see Fig. D.3.). In situations where
retention of radionuclides in the wall of the large intestine is considered, distribution isassumed to be uniform within the mucosa to a depth of 300 µm from the lumenal surface.
(D27) The rectum is divided into an upper region and the anal canal. In the anal canal, theepithelium changes abruptly from simple columnar to stratified. The rectum is treated here aspart of the rectosigmoid for dosimetric purposes.
Table D.4. Reference values for the physiological length of the large intestine (cm) (ICRP,
2007).
Segment
Newborn 1 year 5 years 10 years
15 years
adult
Male
femalemale
femaleRight colon
14
18
23
28
30
30
34
30
Left colon
16
21
26
31
35
35
38
35
Rectosigmoid 15
21
26
31
35
35
38
35
Total length
45
60
75
90
100
100
110
100
Table D.5. Assumed values for the internal diameter of the large intestine (cm) (ICRP, 2007).
Segment
Newborn
1 year
5 years
10 years 15 years
Adult
Right colon
3
4
4.5
5
6
6
Left colon
2.5
3
3.5
4
5
5
Rectosigmoid
1.5
2
2.3
2.5
3
3
D.2.1. Cell kinetics(D28) Stem cells in stratified squamous epithelium, as in the lining of the mouth, thetongue, and the oesophagus, are taken to be located in the basal cell layer, adjacent to thebasement membrane, as assumed for skin (ICRP, 1992). In the stomach, stem cells arethought to be located towards the upper regions of the gastric pits, renewing the epithelial
layer by both upward flow of daughter cells towards the luminal surface of the stomach anddownward flow into the gastric glands (Karam et al., 2003; Modlin et al., 2003).
(D29) In the colon, the site where most intestinal cancers arise, the stem cells are situatedat the very base of the crypts. This has been deduced from a variety of cell kinetic, mutational,
and regeneration studies in mouse models (Potten, 1995). Their position in man is likely to bequalitatively similar. The number of stem cells per colonic crypt in mice has been estimatedto be in the range of one to eight. Colonic crypts in man are around six times as large in all
dimensions as in mice. It is considered that the number of stem cells per crypt may be similar
in man and mouse, but with more transit cell divisions in man.
(D30) The lower third of the crypt constitutes the replicative zone where newly generated
cells undergo two to three more divisions as intermediate cells while they begin theirmigration up the crypt to the luminal surface, where they are shed at the midpoint between
two adjacent crypts. Estimates of the average cell cycle time of proliferative cells have beenreported in the range 34-69 hours, with S phase duration of 9 hours (Potten, 1995). A peak in
the LI was reported in both the human colon and rectum over the range of 10-25 cell
positions from the crypt base (Potten et al., 1992). The maximum LI was about 29% in thecolon and about 22% in the rectum. These figures suggest that in the region of maximum LI,
the cell cycle time may be of the order of 30 hours for the colon and 39 hours for the rectum.
The stem cell cycle time in human colonic crypts was stated as ≥36 hours (Potten, 1995). The
turnover times of the epithelial cells of the colon are similar to those of the small intestine, i.e.

142

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about 6 days for the absorptive cells and goblet cells, and up to 4 weeks for the enteroendocrine cells. Senescent epithelial cells are shed into the lumen.
(D31) The division potential of ISCs is enormous. There may be up to 1,000 stem-celldivisions over the 3-year lifetime of a mouse, and up to 5,000 in man over a lifetime(Marshman, 2002). Several mechanisms have been considered to ensure stem-cell integrity
over this extremely large number of divisions: (a) selective retention of the parental template
DNA strand during cell division (the “immortal strand” hypothesis), demonstrated in theintestine (Potten, 1978); (b) cell cycle checkpoint genes such as p53 and p21 to arrest cell
cycle progression and prompt repair when DNA damage is detected; and (c) removal ofdamaged stem cells by apoptosis after low radiation doses.
(D32) In the past, the number of stem cells per crypt in mice was often inferred from
calculations using dose-survival relationships for whole crypts after high acute doses ofradiation. It was assumed that a crypt would repopulate from one or more surviving stem
cells (called variously colony-forming cells or clonogens). One estimate of the number of
repopulating cells in a mouse colonic crypt was 88 (Tucker et al., 1983) or 105-116 (Tucker
et al., 1991), and even higher in the small intestine. Although it was recognised that somepost-stem cells could also possibly regenerate crypts, the calculated numbers were unrealisticon the basis of the normal cell renewal processes in the crypt. Various technical reasons wereconsidered to be associated with the high calculated numbers, and lower numbers were later
calculated (Cai et al., 1997). At low doses, values of 5-10 clonogenic cells were calculated,
and these were similar to the lower limit to the number of stem cells of about 6 per colonic
crypt estimated from the maximum number of apoptotic cells in the crypt at short times after
irradiation (Potten and Grant, 1998). This was based on the presumption that altruistic cellsuicide would occur in damaged stem cells, and that the number of apoptotic cells would
increase as the dose increased. Then, as the dose increased further, the number of apoptotic
cells would reach some lower limit to the number of stem cells, governed by the increasing
resistance of post-stem cells to apoptosis. Similarities between number of apoptotic cells andclonogens have not been examined in stomach crypts.
(D33) Genetic modification of crypt cell radiosensitivity provides a further test of thecorrelation between numbers of clonogenic/stem cells and apoptotic cells. This has beenaddressed only in the small intestine. In that case (Table D.6.), mice were used which wereseparately null for ATM, p53, or Bcl-2. Low dose-rate radiation was used in order to backextrapolate the dose response curve semi-logarithmically to zero dose, and estimate theclonogen number more accurately. Null-ATM decreased slightly the apoptotic yield,
clonogen number was unchanged, but clonogen radiosensitivity increased 3-4 fold. With nullp53, apoptotic yield decreased, and clonogen number and radiosensitivity tended to decrease.
With null-Bcl-2, apoptotic yield was unaffected, whereas clonogen number andradiosensitivity tended to increase. It was concluded that there were some relative shifts in
apoptotic and clonogenic population sizes caused by these mutations, but it was not possible
to relate these to detailed cell positional data because of the uncertainties involved in thevalues (Hendry, 2002). Hence, the respective roles of apoptosis and mitotic death in theradiation-induced sterilisation of stem cells in the small intestine remain unclear. In addition,
recent data showed that deletion of p21 resulted in protection of crypt stem/progenitor cells
from radiation-induced cell death (George et al., 2009). In the colon, Bcl-2 prevents stem-cell
apoptosis after irradiation, and apoptosis is random throughout colonic crypts. Hence, in thissite, apoptosis does not play a significant role in stem cell killing after radiation, which isprobably mediated by mitotic death following attempted repair involving p53 and p21
checkpoint controls.

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Table D.6. Apoptosis levels and clonogen numbers in small-intestinal crypts in mutant micecompared to those in the wild-type strains FVB and C57 (Hendry, 2002). (Permission needed)(D34) It has been estimated that there are about 5 x 107 crypts in the small intestine in man(Potten, 1995). The length of the human small intestine is about 550 cm, the diameter is about
5 cm, the surface area is about 8,250 cm2, and hence the crypt density is about 6,060 per cm2.
Crypts in the colon are larger than in the small intestine, so the density may be around 4,000
per cm2. The human colon is about 110 cm long, the diameter is about 5 cm, and the surface
area is about 1,650 cm2 which is 1/5 that of the small intestine. Hence, there should be about
6.6 x 106 crypts in the large intestine in man. In a mouse colonic crypt, it is estimated that
there are about 6 (to within a factor of 2-3) stem cells, and probably the same in man. Hence
there may be a total colonic stem cell population in man of about 4 x 107. This is a very roughestimate with large uncertainties.
(D35) There is also the possibility that some daughter cells on the first step towardscommitment and differentiation also might be capable of regenerating all crypt cell
populations, so-called “potential stem cells”, if they can relocate to the stem cell zone afterradiation-induced cell depletion and function as stem cells. This possibility was raised
because of the general discrepancy between many early estimates of high numbers ofclonogens and the low numbers of stem cells expected from cell lineage and kinetic
considerations. The idea is also consistent with the scenario in haematopoietic tissue of its
repopulation by “cord blood stem cells” - cells with lower self-renewal capacity than thenative niche stem cells; nonetheless, they can regenerate depleted marrow quite extensively.
However, in the crypt, later work showed that the higher clonogen estimates could be
accounted for instead by a second mode of clonogen kill at higher doses caused for example
by some radiation damage to the niche (Roberts et al., 2003). Hence, the “potential stem cell”
scenario in the crypt remains uncertain, but if true, it could provide more target cells for
carcinogenesis.
(D36) In the stomach, there are less extensive data. The mucosal surface is 525 cm2. If it
assumed that the parameter values for stomach and colon are similar except for mucosal area,
which in stomach is about 1/3 of that in the colon, the total stomach crypt stem cell
population would be about 107.
(D37) The cell population structure and lineage characteristics for oesophageal epithelium
are under current investigation (Barbera et al., 2014).
D.2.2. Age dependence(D38) Table D.7. lists typical masses for segments of the alimentary tract as a function ofage and gender. Values for adult males and females are rounded central estimates based on
the data of Tipton and Cook (1969). Age-specific estimates for the stomach are based on data
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from Scammon (1919), who collected measurements of stomach mass for 543 subjects in thefirst two decades of life. These data indicate that the growth rate of the stomach in postnatal
life is equal to or slightly greater than that of the body as a whole. Age-specific estimates forthe oesophagus and divisions of the intestines are based on the assumption that the rate of
growth from birth to maturity parallels that of the stomach. Tissue masses are assumed to be
independent of gender to 10 years of age.
Table D.7. Summary of typical values for masses (g) of walls in the GI tract. (Table A.2. in
ICRP Publication 100, 2006).
Component
Newborn 1 year
5 years 10 years
15 years
adult
male
female male
femaleOesophagus
2
5
10
18
30
30
40
35
Stomach
7
20
50
85
120
120
150
140
Small intestine 30
85
220
370
520
520
650
600
Large intestine:
right colon
7
20
49
85
122
122
150
145
left colon
7
20
49
85
122
122
150
145
rectosigmoid
3
10
22
40
56
56
70
70
Total mass

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6010	
  

56

160

400

683

970

970

1210

1135

(D39) Around birth, epithelial proliferation is confined to shallow pockets residing
between the villi of the small intestine of mice. Mature small intestinal crypts appear in thefirst weeks after birth, by a process in which the intervillus pockets invade the wall of thesmall intestine. Similarly, colonic crypts become progressively deeper in early postnatal life.
Intervillus pockets are initially polyclonal, but rapidly become monoclonal through a poorly
understood process of refinement (Schmidt et al., 1988). In order to accommodate the growthof the organ into adulthood, the number of crypt units steadily increases by crypt fission, aprocess in which new crypts form by branching off existing crypts (Totafurno et al., 1987).
(D40) The response of the developing mouse intestine to x radiation using neonates (1 day
postpartum), infants (2 weeks postpartum) and adults (7 weeks postpartum) has been
examined (Miyoshi-Imamura et al., 2010). Irradiated adult small intestinal crypts displayed
two waves of apoptosis. The first wave peaked at 3 hours, followed by a broad wave with apeak persisting from 24 to 48 hours. p53 was expressed during the first wave but not thesecond wave. For the infant small intestine, the intensity of the first wave was approximately
half that of the adult wave, and for the colon, the intensity was even smaller. In neonates,
apoptosis was delayed, peaking at 6 hours for small intestinal crypts, and at 24 hours forcolonic crypts. Although no apoptosis occurred at 3 hours postirradiation in neonates, p53
was present in both the small intestine and colon, owing at least in part to the inability of p53
to increase the level of Noxa, a p53-dependent pro-apoptosis protein, suggesting a
discontinuity in the p53-Noxa-caspase pathway in neonates. By contrast, the induction of p21,
a pro-survival protein, was greater in neonatal cells than in adult cells. Thus, it appears thatthe developing and adult intestine mount different apoptotic responses to radiation.
(D41) As the mammalian GI tract develops from the embryonic gut, it is made up of an
endodermally-derived epithelium surrounded by cells of mesodermal origin. Cell signalling
between these two tissue layers plays a critical role in coordinating patterning andorganogenesis of the gut and its derivatives. Many lines of evidence have revealed that ‘Wnt’
signalling is the most dominant force in controlling cell proliferation, differentiation, andapoptosis along the crypt-villus axis. Wnt mRNA expression in intestinal subepithelial
145

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myofibroblasts and Fzd mRNA expression has been found in both myofibroblasts and crypt
epithelium, as part of the stem cell “niche” (Yen and Wright, 2006). Moreover, there aremany other factors, for example, BMP, ‘homeobox’, ‘forkhead’, ‘hedgehog’, ‘homeodomain’,
and PDGF that are also important to stem cell signalling in the GI tract.
(D42) The effect of ageing has been studied, but only in the small intestine. After highdoses of irradiation, the surviving crypts in old mice (28-30 months of age) were both smallerand fewer in number than in young mice (6-7 months of age). There was also a growth delayof 1-1.5 days in the older mice. Surprisingly, the number of clonogenic cells per crypt wasestimated to be greater in the older mice. These studies indicated important age-related
alterations in the capacity to regenerate the crypts after radiation damage (Martin et al.,1998a). Also, a twofold increase in the level of apoptosis was seen following 1 Gy γ
irradiation in 29-month-old animals, compared to the young and middle-age groups. After 8
Gy irradiation, the level of apoptosis in all age groups was high and the age effect was lesspronounced. The data suggest that stem cells do undergo some functional alteration with age(Martin et al., 1998b). There was also alteration in the level of p53 and p21 expression,suggesting an age-related defect in the capacity to recognise damage and initiate apoptosis or
repair (Potten et al., 2001). It is not known if these effects are also present in the large
intestine.
D.2.3. Cellular features(D43) In human oesophagus and stomach, LRCs have many features of stem cells (longlived, slow cycling, uncommitted, and multipotent), and these have been detected recently ina recognised stem cell niche (Pan et al., 2012). Further analyses of these cells, in healthy andmetaplastic epithelia, are required.
(D44) One of the first suggested specific markers for stem cells was an antibody to Msi-1,
an RNA-binding protein identified as playing a role in asymmetric division control in NSCs,
which appeared to be expressed in very early lineage cells in the small intestine (Kayahara etal., 2003; Potten et al., 2003).
(D45) Also, a large series of studies indicate that the Wnt signalling pathway has a uniqueand central role in the (patho-) physiology of the intestine (Barker et al., 2008). Multiple
secreted Wnt factors are produced by the epithelial cells at the crypt bottom (Gregorieff et al.,2005), potentially generating a morphogen-like gradient of Wnt signals along the crypt–villus
axis. Barker and Clevers (2007) detected several Wnt target genes with a very restricted
expression within crypts. One of these, the Lgr5/Gpr49 gene, was expressed in a particularly
unique fashion. The Lgr5 gene encodes an orphan G protein-coupled receptor, characterisedby a large leucine-rich extracellular domain. It is closely related to receptors withglycoprotein hormone ligands, such as the TSH, FSH, and LH receptors. In situ hybridisation
on small intestinal tissue revealed highly restricted expression at the crypt bottom, also
observed in colon. This expression pattern clearly differed from that obtained with any of theother 80 genes in the Wnt signature. Thus, the Lgr5+ cells in small intestine and colon cells
fulfilled the definition of stemness in displaying longevity and multipotency. The
observations confirmed earlier estimates that each crypt contains approximately six
independent, long-lived stem cells. Counter-intuitively, although these cells are putative stem
cells, they appeared never to be quiescent. Rather, they completed a cell cycle every day (inmice). Tian et al. (2011) employed lineage tracing and cell ablation studies, and reported thatelimination of the rapidly-cycling stem-cell population had no effect on intestinal
homeostasis, leading them to conclude that Lgr5-expressing cells are dispensable. This would
be consistent with the presence of a second lineage pathway.
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(D46) Another study found that the stem-cell marker Bmi1 was expressed in discrete cells
predominantly at cell position 4 above the base of the small-intestinal crypt in mice
(Sangiorgi and Capecchi, 2008). Over time, these cells proliferated, expanded, self-renewed
and gave rise to all the differentiated cell lineages. Ablation of the Bmi1+ cells led to cryptloss. Bmi1-expressing cells were dramatically increased in number following Lgr5-cellablation and they appeared to function as a reserve stem cell pool contributing to intestinal
lineage development via the Lgr5 cell-independent pathway. When the ablation signal was
removed, the Bmi1-expressing cells rapidly gave rise to Lgr5-expressing cells, thereby
restoring the Lgr5-dependent lineage pathway. Also, the induction of a stable form of βcatenin in the Bmi1+ cells was sufficient to rapidly generate adenomas.
(D47) A further study identified a rare population (one cell per 150 small-intestinal crypts)of slowly-cycling stem cells (90-95% in G0, located mostly between cell positions 5-8 fromthe crypt base), marked by mTert expression (Montgomery et al., 2011). The mTert+ cells
were distinct from Lgr5+ cells, but they included a subpopulation of Bmi-1+ cells. The
mTert+ cells were distributed in a pattern along the crypt–villus axis similar to that of longterm LRCs, and they gave rise to Lgr5+ cells. Lineage-tracing studies demonstrated thatmTert+ cells gave rise to all differentiated intestinal cell types, persisted in the long term, andcontributed to the regenerative response following injury. In addition, Takeda et al. (2011)
identified Hopx gene expression as another marker of slowly-cycling stem cells at cell
position 4 in the crypt. Hopx-expressing cells appeared to be present in virtually every crypt,
and could give rise to Lgr5+ cells, and vice versa, indicating the plasticity of the lineages.
(D48) A picture is emerging of a stem cell lineage in the small-intestinal crypt comprisingboth slowly-cycling and rapidly cycling stem cells at about cell position 4, with the latterproducing several progenitor cell lineages. Much less knowledge is available for colonic
crypts, but mTert+ cells and Lgr5+ cells have been detected there.
(D49) Other genetic clonal marking strategies exploit mutation of the X-linked glucose-6phosphate dehydrogenase gene in male mice (Griffiths et al., 1988; Park et al., 1995), of ahypothetical enzyme involved in O-acetylation (Campbell et al., 1996), or of mitochondrial‘cytochrome c’ oxidase (Greaves et al., 2006; Taylor et al., 2003) in human colon. Anotherelegant tracing strategy follows epigenetic changes in gene methylation patterns as a lineage
marker (Shibata, 2008; Yatabe et al., 2001). Other data support the use of doublecortin and
CaM kinase-like-1 (DCAMKL-1), a microtubule-associated kinase, as an intestinal andpossibly colonic stem cell marker (Giannakis et al., 2006; May et al., 2008). On the other
hand, the CD133 marker, which is expressed by normal primitive cells of the neural,haematopoietic, epithelial and endothelial lineages, was reported to be extremely infrequent
in normal colon tissues (Ricci-Vitiani et al., 2007).

6096	
  

D.3. Radiosensitivity

6097	
  
6098	
  
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6100	
  
6101	
  
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6103	
  
6104	
  
6105	
  
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(D50) The radiosensitivity of crypt stem cells is usually measured using clonogenic
radiosensitivity, assuming that stem cells will be capable of regenerating all crypt cell
populations. Clonogenic radiosensitivity measures the most resistant population of clonogens,because as the dose increases crypt numbers start to decline only when the last clonogen has
been killed in a small proportion of the crypts. Hence if there are some more-sensitive
clonogens these will be killed by lower doses and will not contribute to the terminal shape ofthe dose-survival curve, but they will contribute to the threshold dose for crypt killing.
(D51) Crypt clonogens are slightly more resistant to acute doses in the stomach (Fig. D.4.)
and colon (Fig. D.5.) than in the small intestine. In the colon, this is due to the presence of
p53, because in the p53-null mouse, the sensitivities of colonic and jejunal clonogens are the147DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
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6110	
  
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6119	
  
6120	
  

same (Hendry et al., 1997). A possible explanation is the greater involvement of p53 in thelarge than in the small intestine, regarding repair and a G2-phase checkpoint delay.

Fig. D.4. Survival of gastric clonogenic cells exposed to γ rays. Circled points, extrapolated data froma figure published in Chen and Withers (1972). Squared points, deduced using extrapolated values. 1
rad = 1 cGy. Reproduced from Hendry (1979). (Permission needed)

Fig. D.5. Crypt survival curves obtained for the four regions of the small intestine (closed symbols)
and three regions of the large intestine (open symbols). The small intestinal data were obtained 3 days
after irradiation using haematoxylin- and eosin (HE)-stained sections with autoradiography and athreshold of >10 labelled cells and the large intestinal data 5 days after irradiation using vincristine-

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treated animals and a threshold of >3 mitotic cells to define a viable crypt (Reproduced from Cai et al.
1997b). [Note that these are survival curves for whole crypts containing many clonogens, not forindividual clonogenic cells, and hence the presence of the initial plateau portion of the curves. A moredetailed curve in the lower dose region for single clonogenic cells can be found in (Tucker et al.,1983)]. (permission required)(D52) Clonogenic radiosensitivity is lowest at low doses or when the dose rate is low. The
lowest reported value of the α (initial slope) sensitivity parameter for colonic crypt clonogens
is 0.15 Gy-1 (or initial D0 = 5.4 Gy) derived from fractionated dose studies (Tucker et al.,1983). For stomach clonogens, the initial D0 was similar at about 5.5 Gy (Hendry, 1979). GI
clonogenic cells show conventional dose-rate, dose fractionation, and dose protraction effects,
characteristic of cells proficient in repair and repopulation (Figs. D.4. and D.5.; Potten, 1995).
(D53) Generally, the sensitivity of cells that undergo apoptosis is much greater than that ofclonogenic cells (n.b. an exception to this is the rare population of mTert+ stem cells, notshowing apoptosis at the conventional scoring time of 3 hours after 1 Gy or 10 Gy(Montgomery et al., 2011)). Survival curves for apoptically-susceptible cells were generated
for the small intestine by generating a dose-incidence curve over the dose range 0.01-1 Gy,noting the maximum incidence as the dose was increased, and expressing “the maximum
number minus the number observed at any lower dose” as a surviving proportion of thatmaximum. The resultant D0 was 0.1-0.2 Gy (Hendry and Potten, 1982; Potten, 1977), verymuch smaller than the value for clonogens. It was recognised that this high sensitivity wasvery dependent on the maximum value chosen, with the use of higher maximum values
leading to lower sensitivities, but still the big difference remained (Hendry and Potten, 1982).
The sensitivity to apoptosis was independent of dose rate between 0.0027 and 0.45 Gy per
minute (Hendry et al., 1982). In addition, it was shown that when apoptosis in the stem cellzone was induced by an acute dose of 0.5 Gy, the apoptosis-susceptible cells were restored
within 2 days (Ijiri, 1984). This suggests that during chronic irradiation at a very low doserate, continuous deletion of damaged cells and their replacement might occur, but this has not
yet been studied. The apoptosis-susceptible cells are within the stem cell zone in the small
intestine.
(D54) Radiosensitivity of the apoptosis-susceptible cells was greater in the case of highLET neutron irradiation (Hendry et al., 1982). It was calculated that a single radiation trackfrom neutrons would initiate the apoptotic process. For both high and low dose-rateirradiations using low-LET or neutron sources, the distribution of apoptotic cells in the crypt
was similar, indicating that the differential sensitivity of the affected cell populations
remained the same. Further, the ‘clustering’ of apoptotic cells was examined, and found not
to be significantly different from a random distribution. This indicated that there was noevidence for a local ‘bystander’ effect in neighbouring cells, and in the case of the 600 MeV(maximum energy) neutron beam, no evidence for multiple cells hit by nuclear fragmentation‘stars’.
(D55) Cycling CBCCs, another population of stem cells at crypt positions +1-+3 in mousesmall intestine, were shown to be relatively radioresistant, repairing DNA by HR
significantly more efficiently than TA progenitor cells or villus cells. CBCCs underwent
apoptosis less than 24 hours after irradiation (32% ± 2% of total lethality) or mitotic death at
24-48 hours (Hua et al., 2012).
(D56) In the mouse colon, the frequency of radiation-induced apoptosis was highest at thecrypt base, and it declined gradually to more than halfway up the crypt (Pritchard, 2000; Fig.
D.6.). This is consistent with radiation killing of the stem cells at the crypt base as well assome transit daughter cells as they differentiate up the lineage. Also, the frequency of149

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apoptosis in the colon continued to rise as doses increased from 1 to 8 Gy (Fig. D.6.), rather
than almost saturating at around 1 Gy as in the small intestine. This suggests that the transit
cells have increasing resistance to apoptosis. Bcl-w-null animals exhibited more apoptosis
than their wild-type counterparts (Fig. D.6.), indicating the importance of this bcl-2 family
member in causing resistance to radiation-induced apoptosis in these colonic progenitor cells.
(D57) In the mouse stomach, maximum numbers of apoptotic cells were observed in both
antrum and corpus at 48 hours after radiation doses greater than 12 Gy (Przemeck, 2007; Fig.
D.7.). The incidence of apoptosis was much lower than observed in the small intestine orcolon after similar doses of radiation. The highest numbers of apoptotic cells were observed
at cell positions 5–6 in the antrum and cell positions 15–18 in the corpus. These distributions
coincided with the distributions of proliferating cell nuclear antigen (PCNA)-labelled
proliferating cells. In the gastric corpus, the putative stem cell zone and the proliferative zone
are located at the isthmus of the gland. Decreased numbers of apoptotic gastric epithelial cells
were observed in p53-null, bak-null, and bax-null mice, and increased numbers in bcl-2-null
mice, compared with wild-type counterparts 6 and 48 hours after 12 Gy γ-radiation. Detailed
lineage characteristics have not yet been established for stomach crypts.
(D58) Protection of clonogens in the small intestine against killing by radiation has beenachieved by using high levels of certain growth factors and cytokines, such as TGFβ3, IL-11,
and keratinocyte growth factor (KGF) (Booth and Potten, 2001), as well as a variety of otheragents including antioxidants and prostaglandins (reviewed in ICRP, 2012). Most of thesestudies have been directed towards pre-irradiation protective measures for the small intestine
subject to high doses in a therapeutic or accident context. Few studies have focussed on otherregions of the GI tract relevant for carcinogenesis such as stomach and colon. Examples are amoderate radioprotective effect on murine colonic crypts by pre-irradiation treatment using
the aminothiol compound WR-2721 (Ito et al., 1986), and retinoic acid and interferon (Mason,
1994).

Fig. D.6. Cell positional distribution of apoptosis induced by γ-irradiation in colonic intestinal crypts.
The bcl-w wild-type (dashed curves) and homozygously-null (solid curves) mice (four animals ineach experimental group) had received 60Co γ-irradiation 4.5 hours earlier. (c) Midcolon, after 1 Gy.
(d) Midcolon, after 8 Gy. The standard error of the mean is shown for each point (Pritchard et al.,2000). (Permission needed)

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Fig. D.7. A: Time course showing mean (± SE) apoptotic index assessed by HE staining in antrum
and corpus of male CD1 mouse stomach up to 96 hours following 12 Gy γ-radiation (n = 9 at 48 hours,
n = 4–6 at other time points). B: dose response showing mean ± SE apoptotic index induced in antrum
and corpus of male CD1 mouse stomach 48 hours following 1–14 Gy γ-radiation (n = 9 at 12 Gy, n =5 at other doses) (Przemeck et al., 2007). (Permission needed)

D.3.1. Characteristics of single-cell responses(D59) The radiation survival curve for a normal rat intestinal epithelial cell line, IEC-17,
which has features of undifferentiated small intestinal crypt cells, exhibited no initialshoulder and was bimodal (Sierra et al., 1985). The D0 for the first component was about 0.2
Gy and for the second component, representative of about 10% of the cell population, was 5.0
Gy. These values are strikingly similar to the D0 of 0.1-0.2 Gy for the apoptotically-sensitive
cells in vivo in the mouse (paragraph D48), and the initial slope of the survival curves for theclonogenic cells with D0 5.4 Gy (paragraph D47). These results indicate that the bimodal
radiation response observed in vivo can be recapitulated in vitro using a crypt-derived cellline. Extended age of IEC-17 cells in culture (greater than 100 passages) resulted in alteredmorphology, decreased doubling time, increased chromosome number, and loss of anchorage
dependence, all features characterising spontaneously-transformed high-passage IEC-17 cells(DeRose and Claycamp, 1989). These high-passage cells also exhibited a bimodal response to
x-rays, suggesting that the different radiosensitivity of the subpopulations remained
throughout the spontaneous transformation of high-passage IEC-17 cells.
(D60) Single cells have been cloned from disaggregated human and murine normal colonic
crypts (Whitehead RH, 1999), and an epithelial cell line has been established from thecolonic mucosa of protein tyrosine kinase 6 (Ptk6) null mice (Whitehead RH, 2008). PTK6 isan intracellular src-related tyrosine kinase that regulates differentiation in the intestine, and151

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these cells have the characteristics of a stable progenitor cell. When cultured in collagen gel,the Ptk6 null cells form complex organoids, some of which resemble cups of cells, containing
cells with differentiated phenotypes. Also, differential cell surface abundance of ephrin typeB receptor 2 (EPHB2) allows the purification of different cell types from human colon
mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cellswith the longest telomeres and elevated expression of ISC marker genes. Using culturingconditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be
expanded in vitro as an undifferentiated and multipotent population (Jung et al., 2011). Suchcell lines and organoids may be useful for studying aspects of radiation responses at thecellular level.
(D61) Information on the requirements of the crypt niche for stem-cell maintenance andgrowth was reviewed by Shaker and Rubin (2010). It has been shown that mesenchymalderived cells are required for long-term culture of stem cells (Ootani et al., 2009). Using a
microenvironment consisting of an air-liquid interface 3D collagen gel to improveoxygenation, as well as myofibroblasts and stem-cell niche signalling molecules, long-term
culture of intestine and colon was established. Also, Lgr5+ stem cells can be maintained inlong-term culture and differentiate into crypt–villus-like units in the presence of a limited set
of appropriate extracellular growth signals, which normally are derived from the underlying
mesenchyme in vivo (Sato et al., 2009).

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D.4. Mutagenesis

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(D62) A concept of early tumour development has evolved, as described in the UNSCEAR(2000) report and reproduced here. The concept requires a relatively tissue-specific“gatekeeper” gene to be mutated in order for stem-like cells to enter a phase of inappropriate
clonal expansion (Kinzler and Vogelstein, 1996; Sidransky, 1996); this expansion then allows
for the accumulation of further mutations. According to the concept, the accumulation ofother mutations in the neoplastic pathway in the absence of gatekeeper defects will result
only infrequently in the clonal development of recognisable tissue lesions. In essence, thetemporal order of mutational events is likely to be important for productive neoplastic growthwith loss of specific gatekeeper genes as critical early events.
(D63) A key element in this hypothesis, as it relates to colorectal cancer, is that the firstconsistent mutation in tissue lesions should be monoclonal mutation of the APC gatekeeper
gene, which acts as a transcriptional regulator (Nakamura, 1997). In the main, the data
(Kinzler and Vogelstein, 1996) support this, but investigations of the temporal sequence ofgene mutations add considerable weight to the argument.
(D64) Using tumour microdissection and allelotyping methods, the sequence and tempo ofallelic losses in a series of colorectal cancers at different stages of development were
followed (Boland et al., 1995). The principal losses that were tracked were those associatedwith deletion of APC (5q21), p53 (17p13), and deleted in colorectal cancer (DCC) (18q21).
In brief, LOH via allelic loss was not recorded in normal tissue surrounding colorectal
tumours. However, 5q but not 17p losses arose abruptly and consistently at the transition
from normal tissue to benign adenoma; a proportion of adenomas also showed 18q losses.
Losses to 17p occurred equally abruptly and consistently at the adenoma to carcinoma
transition border, and in highly advanced and invasive carcinomas, there was a high level ofallelic variation indicative of clonal heterogeneity due to genomic instability.
(D65) Thus, commencing with APC loss from cells in normal tissue, the development ofcolonic tumours is characterised by abrupt waves of clonal expansion, with p53 loss andchaotic allelic variation being critical watersheds in the evolution of the fully malignant

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phenotype. Considering these and other molecular genetic observations with colorectal
cancer, a temporal model of neoplastic initiation and malignant development has been
proposed (Boland et al., 1995). This is illustrated in Fig. D.8.

Fig. D.8. A model of the sequence of genetic events in neoplastic development in the human colon(Boland et al., 1995; UNSCEAR, 2000). The colon is one of the clearest examples of elucidation of
the mutational steps involved in carcinogenesis. Initial mutations of LOH at the APC locus of acolonic epithelial cell produce dysplastic crypts followed by adenoma development involving k-ras
mutation and DCC loss. Loss of p53 from advanced adenoma marks the transition between benign
and malignant disease characterised in turn by the development of genomic instability, multiple genelosses and invasive behaviour/metastasis to regional lymph nodes. (permission needed)(D66) Mutation/loss of the tumour-suppressor gene APC has for some time been believed
to be a critical early event in the development of human colon cancer. Up to about 70% of
early colonic adenomas show apparently monoclonal structural/functional loss of this gene
(Powell et al., 1992), and a critical role in tumour initiation seems likely (Boland et al., 1995).
With use of a mouse (Min) model of intestinal carcinogenesis, this view of monoclonal
tumour initiation has been strengthened. In essence, aberrant crypts, the earliest intestinal
lesions detectable microscopically, have been microdissected from Min mice and shown to be
monoclonal with respect to Apc loss (Levy et al., 1994; Luongo et al., 1994). On the other
hand, it has been found that a large proportion of intestinal adenomas in the GI tracts ofhuman FAP patients, who were also XO/XY in genotype and therefore mosaic for the Ychromosome, was apparently polyclonal. In this study, polyclonality was judged by thepresence within single adenomas of a mixed population of cells with respect to the Ychromosome sequence. By this measure, up to 76% of adenomas were polyclonal. However,
early Y-chromosome loss and field effects creating tumour clustering and collision(UNSCEAR, 2000) might contribute to this finding.
(D67) Tumour clustering may also explain data on the apparent polyclonality of a
proportion of spontaneously arising intestinal adenomas in Min mice as assessed by genetic
features other than Apc loss (Dove et al., 1998). Thus, polyclonality may be acquired during
adenoma development rather than arising de novo at the time of initiation. For example,

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fusion of independent Apc-deficient microclones may allow for cooperative growth. These
data illustrate some of the problems that remain in resolving the early molecular events andcomplex cellular interactions of tumour development. In spite of these uncertainties, there
remain experimental data on intestinal tumourigenesis that forcibly support monoclonality for
induced neoplasms (Griffiths et al., 1989).
(D68) Based on investigation of the earliest colonic tissue alteration in FAP patients,
Boman et al. (2008) presented the hypothesis that initiation of colorectal cancer by APC
mutation is mediated by dysregulation of two cellular mechanisms. One involves
differentiation, which normally decreases the proportion (proliferative fraction) of colonic
crypt cells that can proliferate; the other is a cell cycle mechanism that simultaneously
increases the probability that proliferative cells are in S phase. In normal crypts, stem cells at
the crypt bottom generate rapidly proliferating cells, which undergo differentiation while
migrating up the crypt. Modelling studies of normal crypts suggest that these transitions are
mediated by mechanisms that regulate the proliferative fraction and the S-phase probability.
In FAP crypts, the population of rapidly proliferating cells is shifted upwards, as indicated by
the LI. Analysis of FAP indicates that these transitions are delayed because the proliferative
fraction and S-phase probability change more slowly as a function of crypt level. This leads
to expansion of the proliferative cell population, including a subpopulation that has a low
frequency of S-phase cells.. Boman et al. (2008) reported that stem cells (or cells having highstemness) are proliferative cells with a low probability of being in S phase. Thus, it was
concluded that dysregulation of mechanisms that control proliferative fraction and S-phaseprobability explains how APC mutations induce system cell overpopulation at the cryptbottom, shift the rapidly proliferating cell population upwards, and initiate colon
tumourigenesis. It has also been shown that Lgr5+ stem cells, which represent about 5 to 10%of the cells in mouse intestinal adenomas, generate additional Lgr5+ cells which fuel adenoma
growth, as well as all other adenoma cell types (Schepers et al., 2012). The Lgr5+ cells wereintermingled with Paneth cells near the adenoma base, a pattern reminiscent of thearchitecture of the normal crypt niche.
(D69) A model for colonic crypt organisation, patterns of stem cell divisions, niche
succession and clonal conversion (Fig. D.9.), was described by Humphries and Wright (2007).

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Fig. D.9. Colonic crypt organisation, patterns of stem cell divisions, niche succession and clonal
conversion. Panel A. A diagrammatic representation of the colonic crypt. Stem cells are housed in thebase of the crypts where they communicate with the niche cells — the pericryptal myofibroblasts,
which are outside the crypt but communicate by cell signalling. Stem cells feed the TA compartment,
where most cell production occurs. This portion of the crypt is also thought to house the committedprogenitor cells: cells committed to one or more cell lineages. Panel B. Patterns of stem-cell divisions(a) shows an asymmetric stem-cell division, in which a stem cell gives two daughters, one remaining
in the niche as a stem cell and the other committed for differentiation, sometimes called q divisions. If
all stem cells underwent such asymmetric divisions, stem cells would remain immortal: their progeny
remain within the niche. However, several strands of evidence indicate that other stem-cell divisions
occur: symmetric divisions, producing either two stem cells — so-called p divisions (b) — or twocells destined to differentiate — r divisions (c). If the incidence of r divisions or apoptosis is >0, thena stem cell will eventually come to dominate the niche. Thus, in a stem-cell niche, all three types ofstem cell divisions are found. Panel C. Niche succession. (a) shows the colonic crypt niche, housing
the stem cells. In (b), a stem cell has developed a mutation (blue) — say in the cytochrome c oxidase
gene. By asymmetric division, a clone of mutated cells in the above dividing transit compartment
develops from this mutant stem cell, shown in (c). At this stage, the clone could be lost if the mutant
stem cell should undergo apoptosis or a symmetric r division. However, if adjacent stem cells undergo
apoptosis or an r division, the mutant stem cell may expand through p divisions to colonise the niche,as shown in (d). Finally, the mutant stem cell comes to dominate the niche (e). Of course, if themutation occurs in a tumour suppressor gene or an oncogene rather than a housekeeping gene, which
confers a selective advantage, then niche succession would occur more rapidly. Panel D. Clonal
conversion: the progeny of a mutant stem cell replaces all other cells in the crypt (Humphries andWright, 2008). (Permission needed)(D70) Cancers are generally considered to originate from stem cells, i.e. cells that possess
unlimited reproductive capacity. These are transformed by carcinogenic agents so that theirdifferentiation patterns are altered in such a way that cell renewal predominates overdifferentiation, leading to growth of an abnormal cell population. It has also been consideredthat the carcinogen target cells are the actual stem cells in small intestine which are protected

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from tumour induction in the small bowel by an altruistic suicide of cells bearing
carcinogenic lesions, while those in the large bowel are not protected by this altruistic cellsuicide and hence can initiate cancers (Potten et al., 1992). The altruistic apoptosis in thelarge bowel is prevented by the expression of the survival (anti-apoptotic) gene bcl-2, which
is not expressed significantly in the small intestine (Merritt et al., 1995).
(D71) In contrast, it was suggested that tumours in the human colon may originate in cellson the intercryptal plate rather than, or in addition to, stem cells at the base of the crypt (Shihet al., 2001). That study indicated that most early neoplastic lesions of the colon containdysplastic cells only at the orifices of crypts and on the luminal surface between crypts.
Analysis showed loss of the APC gene and high expression of β-catenin in such dysplastic
cells but not in cells with normal appearance within the crypts. Mutations in the APC geneare the earliest genetic alterations in the genesis of colorectal tumours, and appear to berequired to initiate clonal evolution, involving overexpression of β-catenin (Fodde et al.,2001).
(D72) This suggestion of target cells on the lumenal surface is contentious. In normal tissue,
differentiated epithelial cells on the intercryptal surface would have a very limited lifespan ofa few days, destined to be lost in the intestinal lumen in the normal process of cell renewal.
To develop into a tumour, these dysplastic cells would need to escape this process to allow
time for progression to malignancy, involving a sequential number of mutational events(Goyette et al., 1992; Vogelstein et al., 1988). Preston et al. (2003b) reported a detailed
examination of early lesions from patients who had undergone colectomies for FAP and alsoof sporadic colorectal adenomas, from which they concluded that adenomas originate insingle crypts and grow initially by crypt fission. Only in established sporadic adenomas wasthere evidence of growth downwards into adjacent crypts. There has been considerable
debate about what happens next in the progression to an established adenoma: whether
dysplastic cells spill over to invade and colonise the territory of adjacent crypts — a topdown process; or whether the monocryptal adenoma spreads by fission, in a manner
described to account for field cancerisation in the colon — the bottom-up proposal. Although
it is difficult to obtain definitive dynamic proof in humans, clonal expansion by crypt fission
appears to be the prominent mode of spread of an adenomatous crypt, and is now generally
accepted as the most important mechanism for clonal expansion in early adenomas(Humphries and Wright, 2008; Renehan, 2002; Zeki et al., 2011).

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D.5. Target cells and radiation protection

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(D73) In the ICRP (2006) report on the human alimentary tract, doses were calculated tothe estimated position of the stem cells. However, in considering uncertainties in dose
estimates, the possibility that cells higher in the crypts may also be targets was addressed,
including the extreme possibility of target cells on the lumenal surface. Doses were calculated
separately for the mucosal layer of each region of the human alimentary tract (ICRP, 2006).
For penetrating radiations, it was considered reasonable to use the average dose to the wallsof each region as a measure of the dose to the mucosal layer. For non-penetrating α and β
particle emissions, the dose is dependent on the assumptions made regarding the location oftarget cells for cancer induction. For each region of the alimentary tract, the target was taken
to be the stem cells that are located in the basal layer of the stratified squamous epithelia of
the oesophagus and within the crypts that penetrate the mucosal layer in the stomach andlarge intestine.
(D74) Uncertainties were illustrated for the specific case of doses to the colon, because
colon doses are generally the major contributors to relevant alimentary tract doses concerning

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carcinogenesis. Although it is generally accepted that the stem cells in the base of the crypts
are the targets for colon cancer induction, some uncertainty was raised by observations ofdysplastic cells on the lumenal surface of the colon between apparently normal crypts (Shihet al., 2001), although this finding was challenged by other authors (Preston et al., 2003c).
Thus, as well as uncertainties in the depth of the crypts and hence the depth of the stem cells,
there is also uncertainty as to whether it is only the stem cells that should be regarded astargets.
(D75) Table D.8. compares colon doses for different assumptions of target cell location,
normalised to the default assumption that they form a continuous layer at a depth of 280–300µm from the lumenal surface of the colon. Thus, uncertainties in the depth of the crypts andhence the depth of the stem cells, represented by Columns 2 and 3, result in differences ofabout ±10% for 115Cd and smaller differences for the other examples considered. For 234U
and 239Pu, there is no dose to the colon wall from activity in the lumen, and thus no changewith differing assumptions regarding stem cell depth. Similarly, widening the target toinclude cells at higher positions up the crypts (200–300 µm), and thus increasing the mass oftarget tissue, results in a maximum change in colon dose of about 10% for 115Cd. The extreme
assumption that the target may include all epithelial cells from the base of the crypts to theluminal surface (0–300 µm) results in a larger increase in dose. The increase by factors of
about 1.5 for 234U and 3 for 239Pu is relative to the dose to the colon resulting from activity
absorbed to blood. However, these increases in colon doses from 234U and 239Pu will makenegligible differences to committed effective doses, which are dominated by contributions
from doses to tissues and organs from activity absorbed to blood.
Table D.8. Differences (%) in dose coefficients (h) for the colon, compared with the default
casea, resulting from considerations of target depth in the mucosa, considering ingestion by
adult males. Table reproduced from Table 8.5. in ICRP (2006).
Nuclide
55

Fe
Fe
90
Sr
106
Ru
115
Cd
234
U
239
Pu
59

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a

Assumed location of the target region depth from lumen (µm)
220-240
340-360
200-300
0-300
0%
0%
0%
0%
1%
1%
1%
6%
7%
6%
5%
21%
3%
2%
2%
8%
13%
9%
9%
38%
0%
0%
0%
148%
0%
0%
0%
317%

Default case assumes a target depth of 280–300 µm.

(D76) Regarding potential committed lineage cell targets for carcinogenesis, there also
may be evidence of long-lived committed progenitor cells in the human colon: crypts can be
identified where the cyclooxygenase (COX)-mutated clone is present well above the stemcell zone (Humphries and Wright, 2008). This might represent a clone which originated in thestem-cell region but now is in transit to be lost eventually at the top of the crypt by extrusion;
but it may also represent a mutation occurring in such a committed progenitor cell. The
dynamics of this process are not yet known, but interpolation from the rarity of such events
suggests that they last for a long time. This raises the possibility that the cell which undergoes
the first mutation and selection event is not the stem cell, but a committed progenitor cell.
Although there is no direct evidence for this, particularly in humans, this occurrence would
explain several common observations: tumours arise from the colon that are composed
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predominantly, or exclusively, of mucin-secreting cells, endocrine cells or even Paneth cells(Humphries and Wright, 2008).

6466	
  

D.6. Summary

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(D77) ICRP radiation risk weighting factors in the alimentary canal are highest in stomach
and colon (0.12 each), lower in oesophagus (0.04), and extremely low in small intestine (nearzero, classified in remainder tissues). The target cells for carcinogenesis are considered to be
predominantly the mucosal stem cells, situated in the basal layer of the oesophageal
epithelium or in crypts (gastric pits) of the stomach (at 1/3 of the crypt depth from the top),small intestine (at cell position 4 from the base), and colon (at the crypt base). The locationinfluences the risk of short-range radiations from radionuclides by varying extents. The
possibility that stem-cell daughter cells are also target cells for carcinogenesis remains
unclear, and this could also influence risk variously.
(D78) The cytotoxic response of most of the normal stem cells to increasing doses ofradiation is bimodal. Low doses of a few cGy induce apoptosis in the stem cell zone in thesmall intestine, compared to throughout the crypt in the colon. In the small intestine, thiseffect is considered protective by deleting injured stem cells. In the colon, anti-apoptotic bcl2 is expressed which prevents this. After higher doses, some stem and other progenitor cells
are reproductively sterilised, and after doses above 8 Gy, whole crypts are sterilised. The
increasing proportion of sterilised crypts observed as dose is increased, enables deduction of
the radiosensitivity of clonogenic cells. The radiosensitivity is slightly higher in the small
intestine than in the colon, but this difference is due to the differential effect of p53 in the twosites.
(D79) When high cytotoxic doses of radiation are used, crypt clonogenic cells show
marked sparing effects when the radiation dose rate is reduced, the dose is fractionated, or thedose is protracted so as to allow repopulation from surviving stem cells. When low doses areused, most damaged stem cells are removed by apoptosis and replaced, except the rare mTertexpressing cells and those stem/clonogenic cells in the colon expressing bcl-2. The recent
findings of some stem cells that are either slowly cycling or in G0 provides a mechanism forthe multiple mutation and multistage development of malignancy. The most plausible
pathological mechanism is the fission and expansion of dysplastic/aberrant crypts leading toadenomas.

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ANNEX E. LUNG STEM CELLS

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E.1. Lung cancer

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E.1.1. Occurrence of lung cancer(E1) Lung cancer is the most common cancer world-wide accounting for 1.6 million newcases annually (resulting in about 19% of all cancer deaths) (Ferlay et al., 2010; ICRP, 2007).
While smoking is the major cause for about 80% of lung cancer cases, there are a significant
number of lung cancers that occur in never smokers. Specific mutations in the tyrosine kinasedomain of EGFR are implicated in lung cancers arising in non-smokers indicating that there
are significant non-smoker related exposures leading to lung cancer (Lynch et al., 2004; Paezet al., 2004). Indeed, non-smoker lung cancers are now the seventh leading world-wide causeof cancer related deaths, and more common than cancers of cervix, pancreatic or prostate(Parkin, 2002).
(E2) The most common form of lung cancer in never-smokers is ADC (Brownson et al.,
1998; Dibble et al., 2005; Kabat and Wynder, 1984; Ko et al., 1997; Muscat and Wynder,
1995; Radzikowska et al., 2002; Stockwell et al., 1992; Toh et al., 2006; Yu et al., 2006;
Zhong et al., 1999), and the reason for this type of lung cancer is not known. Lung cancer innever-smokers occurs more frequently in women and varies considerably by geographicalregion (Brennan et al., 2006; Brownson et al., 1998; Cerfolio et al., 2006; Dibble et al., 2005;(Lynch et al., 2004; Paez et al., 2004; Parkin et al., 2002). Du et al., 1996; Gorgoulis et al.,2005; Kabat and Wynder, 1984; Koyi et al., 2002; Kreuzer et al., 1999; Muscat and Wynder,
1995; Radzikowska et al., 2002; Shimizu et al., 1984; Toh et al., 2006; Wakai et al., 2006).
Paradoxically, the rate of lung cancer in male never-smokers is less variable, suggesting agreater contribution of risk factors other than smoking in females, including hormonal
regulation. Irrespective of gender, there are many environmental risk factors that areassociated with lung cancer in never-smokers including asbestos, chromium, arsenic, nickel,
indoor radiation (radon) and outdoor air pollution exposures (Alberg et al., 2005; Brennan etal., 2006; Subramanian and Govindan, 2007).
E.1.2. Multistep process of lung cancer pathogenesis(E3) Lung cancer develops in a series of multiple steps extending over years. Conceptually,
this is divided into 3 phases: initiation (accumulation of genetic and potentially epigenetic
changes); promotion (selective growth of cells with these changes over normal cells); andprogression (development of invasive cancer and the metastatic phenotype). It is important toconsider the concept of “field carcinogenesis” where clones of multiple independent
preneoplastic lesions occur within the lung stochastically or after exposure to carcinogens
including radiation.
(E4) The above steps are associated with numerous genetic and epigenetic lesions causedby exposure to cigarette smoke, carcinogens including radiation, and inter-individual
variation in risk of developing lung cancer. It is considered that multiple genetic andepigenetic abnormalities must occur before a lung tumour becomes clinically evident (Minnaet al., 2002; Sekido et al., 2003; Zochbauer-Muller et al., 2002). Alterations can occur at thegenetic level through chromosomal changes such as large or small gains and deletions, and at
the nucleotide level, or through epigenetic changes such as DNA methylation. There havebeen many detailed studies characterising the number and type of both genetic and epigenetic

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changes, as well as the relationship to smoking and geographical and gender influences which
help to provide quantitative estimates of the number of steps involved (Girard et al., 2000;
Toyooka et al., 2004; Toyooka et al., 2001; Toyooka et al., 2003; Zochbauer-Muller et al.,2001). The genetic changes in lung cancer include activation of oncogenes and other growthpromoting genes, and inactivation of tumour suppressor genes (Hanahan and Weinberg,
2000). Also, it will be important to test for the other potential changes, as well as forvariables such as gender, race, and environmental exposure differences.

6551	
  

E.2. Radiation induction of lung cancer in humans

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(E5) Compared with smoking, radiation is much less potent, but it does contribute to thedevelopment of lung cancer. The lung is one of the most sensitive tissues to induction ofcancer and ICRP gave a tissue weighting factor of 0.12 to the lung (ICRP, 2007). Twosources of information for radiation related lung cancer cases come from A-bomb survivors
with external radiation, and miners and the public with internal exposures to radon.
(E6) The A-bomb survivor data include a large cohort, long follow-up and relatively good
dosimetry. The dose response for lung cancer incidence among A-bomb survivors is linear
for lung doses up to 1.5 Gy with a gender-averaged ERR of 0.81 per Gy (Preston et al.,2003a). The ERR for females is larger than for males by a factor of 4-5.
(E7) Radon is an important environmental risk factor known to be associated with lungcancer, and UNSCEAR assembled publications to assess the effects of radon in animal model
systems and in humans (UNSCEAR, 2006). The assembled studies implicated radon incancer of the lung but not that of other tissues. A pooled analysis of 13 European case-control
studies demonstrated a linear dose-response for lung cancer incidence against residential
radon concentration up to 1,200 Bq/m3 with an ERR of 0.16 at 100 Bq/m3 (Preston et al.,2003a). When assessed separately for cancer types, ERRs per 100 Bq/m3 were 0.31, 0.06 and
0.04, for small-cell lung cancer (SCLC), ADC and squamous cancer, respectively.
(E8) ICRP Publication 115 (2010) on radon and progeny examined the risk for lung andother cancers after either residential exposure or exposures of underground miners. A
compelling case was made for the induction of lung cancer and leukaemia as a result of
residential and underground mining exposures, but not for other solid tumours. For
underground miners, based upon recent analyses of Czech and French mining cohorts (where
exposures were considered low, and where exposure estimates were of good quality), a
significant association between lung cancer and low-level radon exposures was describedwith ERRs of 2.0 and 3.4 per 100 working level months (WLM) (Tomasek et al., 2008;
Vacquier et al., 2009). Most studies of underground miners do not take into account smoking
status. However, Publication 115 noted that the estimated ERR for smokers is generally
larger than for non-smokers. One study suggested that there was a relationship between theincreasing ERR per 100 WLM and the increasing number of cigarettes smoked daily(Villeneuve et al., 2007). Publication 115 summarised the estimates of ERR per 100 WLM.
Although there are variations in study populations, data in three studies used for the analysis(Lubin et al., 1995; UNSCEAR, 1999) were highly concordant, with ERRs per WLM of 0.49,
0.59 and 0.59, respectively. These studies used the largest numbers of cohorts and the largest
numbers of miners. At sufficiently high exposures, an inverse effect of exposure rate was also
observed (Lubin et al., 1995) (see below).
E.2.1. Effects of age at exposure and age since exposure160

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(E9) Analyses of the lung cancer incidence among A-bomb survivors demonstrated thatlung is one of the few sites for which the ERR increased with increasing age at exposure(Preston et al., 2003b). Thus, the survivors exposed at the age of 40 years showed a higher
lung cancer risk than those exposed at the age of 10 years. This is interesting since other solidcancers exhibit the opposite age-at-exposure effect where a younger age-at-exposure gives ahigher ERR. Part of this lung-specific age-at-exposure effect could possibly be biased by achange in the smoking habit. Smoking was considerably less prevalent among those witholder ages at the time of bombing than among the younger people. Indeed, an analysis for a
sub-cohort of A-bomb survivors with information on smoking did not reproduce an increaseof the ERR with age at exposure (Preston et al., 2003b). A high ERR of lung cancer afterradiation exposure decreased with attained age (Preston et al., 2003c). In contrast, EAR
increased drastically with attained age, and decreased with age at exposure.
(E10) The BEIR VI (1999) analysis found a decrease of the ERR with attained age for lungcancer risk after exposure to radon. In the age group 75 years or older, the ERR was lower
than in the age group younger than 55 years by a factor of about four (Preston et al., 2003b).
The pooled study of Czech and French miner cohorts also demonstrated the dependence of
the ERR on time after exposure with a decrease in the ERR by 55% per decade (Preston et al.,2003b). Thus, the trend of decreasing ERR by attained age since exposure is shared in lungcancers associated with both low-LET A-bomb radiation and high-LET radon-related αparticle radiation.
E.2.2. Effect of smoking(E11) Smoking interacts multiplicatively with radiation in the case of radon. Thus, the RRof radon for non-smokers is 0.16 per 100 Bq/m3. While smokers exhibit roughly a 20 times
higher incidence of lung cancer than non-smokers, their RR for radon is the same as that of
the non-smokers (Preston et al., 2003b). As for low-LET A-bomb radiation, the effect ofsmoking was once thought to be additive (Preston et al., 2003b). However, recent re-analysesof the LSS cohort has indicated the generalised interaction model in which the joint effect ofradiation and smoking is super-multiplicative for light to moderate smokers with less than apack of cigarettes a day, while the effect was additive or even sub-additive for heavy smokers(Furukawa et al., 2010).
E.2.3. Data on different external/internal/high-LET/dose-rate exposures(E12) Radiation carcinogenesis is assumed to operate as though there is no threshold dose.
However, quantification of that risk for any cancer is problematic, and there are very large
uncertainties. Mullenders et al. (2009) described current biophysical modelling and some of
the challenges for researchers examining low dose or protracted radiation exposures(Mullenders et al., 2009). Up to a decade ago, the two main sources of information for lungcancer risk from exposure to ionising radiation for humans were A-bomb survivors, whowere exposed to external radiation, and uranium miners, whose main exposure pathway wasinhalation of radon. In the past years, our knowledge on lung cancer risk after exposure toionising radiation was improved considerably by studies of lung cancer related to residential
radon, and by studies of the Mayak workers, who were exposed to plutonium and externalradiation.
(E13) A-bomb survivors. The latest analysis of site-specific cancer incidence among Abomb survivors from Hiroshima and Nagasaki includes 112,952 cohort members at the startof the reporting period on 1 January 1950 (Preston et al., 2007). At the end of the reporting161

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period (31 December 1998), 52% of them were still alive. In total, 1,759 cases of lung cancer
were registered, 43% of them among females. Histological confirmation was obtained for
73%. Similarly to other cancer registers in Japan, the lung cancer incidence rate among Abomb survivors increased from 25 (7.5) per 105 males (females) in the period 1975-1979 to
37 (11) per 105 males (females) in 1995-1999. About 117 of the lung cancer cases were
estimated to be related to radiation exposure from the A-bomb detonation. This amounts to
14% of all cancer cases that were attributed to radiation exposure. The role of smoking in thiscohort is limited to one analysis to date (Pierce et al., 2003). Lung cancer among A-bomb
survivors is significantly associated with lung dose with no evidence of non-linearity over the0- to 2-Sv dose range (Preston et al., 2007). Lung cancer incidence has a similar ERR per
dose as mortality (Preston et al., 2003b). The ERR for females is larger than for males by afactor of 2 for mortality and a factor of 5 for incidence. The latter value is higher than for allother site-specific cancer incidence data among A-bomb survivors.
(E14) Animal studies of lung cancer from external radiation sources have compared γirradiation with fast or fission neutron exposures at varying dose rates (Ullrich, 1983; Ullrich
et al., 1979; Ullrich et al., 1977; Ullrich and Storer, 1979; Upton et al., 1970). While thesestudies examined the neoplastic process overall, data specific to lung cancers were generated
across different strains of mice. RBE values of 15-20 were determined for acute neutron
exposures while chronic neutron exposures were less effective at low total doses but more
effective at high total doses. Subsequently, Storer et al. (1988) determined that the RR forlung cancer was not significantly different between humans and mice.
(E15) A comparison of neutrons and γ-rays in a rat model determined an RBE of 30-40 at aneutron dose of 0.1 Gy and more than 50 if the neutron dose was reduced to 0.016 Gy(Lafuma et al., 1989). Subsequently, both a two-step and a two-mutation model were
proposed as models for lung tumours in rats exposed to radon (Heidenreich et al., 1999;
Moolgavkar et al., 1990). In both models, it was assumed that radiation is associated with thefirst step and less so with the second step. In addition, an inverse dose rate effect was
described for neutrons (Heidenreich et al., 1999). Other animal studies that focus on the
spectrum of mutations or other molecular events that may drive lung cancer induction after
the inhalation or radon or other isotopes are described below.
(E16) Miners. In 1999, the BEIR VI Committee gave a comprehensive overview andanalysis of data on lung cancer related to exposure to radon (1999). BEIR VI based its risk
models on analyses of combined data from 11 miner studies. Data were available for 60,606
miners including 2,674 lung cancer cases. BEIR VI developed sophisticated models to
describe effect modification.
(E17) ICRP Publication 115 (2010) tabulated the lifetime excess absolute risk (LEAR)
generated from models that included multiple cohorts of miners. Tomasek et al. (2008)
derived similar LEAR values using the risk model from Publication 65 while using
background reference rates from Publication 60 and Publication 103. However, if the BEIR
VI model including time since exposure (TSE), age and concentration was used, the LEAR
nearly doubled from 2.8 x 10-4 to 5.3 x 10-4 per WLM. By using the Czech-French risk model(Tomasek et al., 2008) that considered to have the best quality of exposure assessment, theLEAR dropped to an intermediate value of 4.4 x 10-4 per WLM. Publication 115 argued thatthe increase in LEAR is the result of the consideration of low dose rate exposures and theincreased ERR per WLM of recent studies as mentioned earlier. As described in Publication
115 (2010), other independent calculations confirmed the BEIR VI and Czech-Frenchmodelling, while inclusion of analyses of more recent single groups generates a range ofLEAR of 3-7 x 10-4 per WLM. Publication 115 notes the sensitivity of such models regarding
sex and ethnic background, for which background lung cancer risks can vary. The
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Commission now recommends a LEAR of 5 x 10-4 per WLM for radon and progeny-induced
lung cancers, instead of the Publication 65 value of 2.8 x 10-4 per WLM.
(E18) BEIR VI used models in which the ERR depends linearly on exposure. For miners,
exposure was specified by a quantity related to the cumulated concentration of short-lived
progeny of radon at the workplace using the unit ‘WLM’. Assuming some typical conditions,an exposure rate of miners of 1.6 WLM year-1 corresponds to the exposure by residential
radon with a concentration of 400 Bq m-3. Thus a 30-year exposure at this level corresponds
to about 50 WLM. For exposures up to 50 WLM, no dependence of the risk on exposure rate
was detected. At higher exposures, however, BEIR VI found an inverse dose-rate effect, i.e.,
for the same exposure level, a lower exposure rate or a longer lasting exposure causes ahigher lung cancer risk.
(E19) The analysis of the German Wismut miner data was also based on a linear
dependence of the lung cancer risk on exposure (Grosche et al., 2006). The resulting ERRper-exposure value of 0.21 (95% CI: 0.18, 0.24) per 100 WLM was lower than the BEIR VI
result by a factor of more than three. The presence of an inverse exposure-rate effect at highexposures was confirmed. After adjustment for smoking and asbestos exposure, the result for
the ERR per exposure in the Wismut case-control study was even lower than in the cohort
study by a factor of two (Brüske-Hohlfeld et al., 2006). An exposure-rate effect was notdetected in that study.
(E20) UNSCEAR (2009) calculated a weighted mean average ERR per exposure from theresults of 9 large miners studies (excluding the Wismut cohort). The studies included a totalof 3,380 lung cancer cases. An estimate of 0.59 (95% CI: 0.35, 1.0) per 100 WLM was
obtained.
(E21) Residential radon. Darby performed a collaborative analysis of individual data from
13 case-control studies in Europe (Darby et al., 2005). Before starting most of the single
studies, considerable efforts were made to have comparable study designs. The analysis
included 7,148 persons with lung cancer and 14,208 without lung cancer. The mean
concentrations of residential radon were assessed for the period of 5 to 34 years before thediagnosis of, or death due to, lung cancer, and for the corresponding time period for thecontrol persons. The strengths of the study include the large number of lung cancer cases, thehomogeneity of the study designs, and the detailed information on smoking behaviour
including analyses performed using stratification by smoking history. Relative to a radon
concentration of 0 Bq m-3, the ERR was significant in the radon concentration category 100199 Bq m-3, and in the two categories with radon concentrations exceeding 400 Bq m-3. The
estimate of the ERR per radon concentration was 0.08 (95% CI: 0.03, 0.16) per 100 Bq m-3.
The relation remained significant when the analysis was limited to measured radon
concentrations smaller than 200 Bq m-3. Correcting for a bias due to uncertainties in themeasured radon concentrations doubled the ERR-per-radon-concentration estimate.
(E22) There was no evidence for a threshold or a non-linear response to the radon
concentration (Darby et al., 2005). The upper 95% CI for a threshold was 150 Bq m-3. For
males, the best estimate of the ERR per radon concentration was higher than that for females
by a factor of four, although it was not significant. The results of a North American poolingstudy (Krewski et al., 2005) on the ERR per radon concentration are consistent with theEuropean pooling study, when uncertainties in measured radon concentration were taken into
account. Publication 115 (2010) described the pooled analysis of three residential casecontrol studies including Europe, North America and China, and estimated that the risk oflung cancer increased by at least 8% for radon concentrations of 100 Bq/m3, with an ERR of
0.16 per 100 Bq/m3 increase. These values take into account an exposure period of at least 25
years. Furthermore, these values are considered to be robust enough on their own to provide
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protection of the public without the use of dose conversions based on underground miner
exposures as was suggested in ICRP (1993).
(E23) Mayak workers. The Mayak Production Association in Southern Urals wasestablished to produce plutonium for the nuclear weapons of the former Soviet Union.
Sokolnikov et al. (2008) evaluated lung cancer in a cohort of 17,740 Mayak workers, whowere initially hired during the period 1948-1972. Among them, 3,924 were exposed toexternal radiation only, for 5,572 the lung dose from α-radiation was assessed based on
measurements of plutonium in the urine, and 8,244 were potentially exposed to plutonium but
not monitored. By the end of 2003, 681 lung cancer deaths had occurred. External radiation
was related to 8.6% of cases, while internal radiation from incorporated plutonium was
related to 29% of cases. For external exposures, excess risks depended linearly on lung dose
without any evidence for a threshold (Jacob et al., 2009; Sokolnikov et al., 2008). The ERR
per dose was compatible with the corresponding result for A-bomb survivors.
(E24) For plutonium exposures, Sokolnikov et al. (2008) found, in a non-parametricanalysis of lung cancer mortality among Mayak workers, no excess risks for lung doses
smaller than 100 mGy, a non-significant excess risk in the dose range of 100-200 mGy, andsignificantly increased risks for larger doses. In a recent analysis of a sub-cohort of maleworkers with information on smoking and alcohol consumption, the description of the data bya linear ERR model was significantly improved by introducing a dose threshold in the rangeof 100-200 mGy (Jacob et al., 2009). An even better description was obtained by a model ofcarcinogenesis, in which the dose rate dependences of the initiation rate and of thehyperplastic growth rate had a threshold in the order of 10 mGy year-1.
(E25) Summary. For external exposures, there is no evidence for a deviation from an LNT
description of the excess lung cancer risk versus lung dose, and this is also the case with moststudies on internal exposures to α-radiation. However, a recent analysis of lung cancer
mortality among Mayak workers showed evidence for a threshold in the dose response atabout 100 mGy, or in the dose-rate response at about 10 mGy year-1. A weighting factor of 20
was proposed for radon by the ICRP (2007). However, the direct measurement of radiationdose given by inhaled radon to the target cells in the respiratory tract was not considered
possible. Publication 115 (2010) reviews effective dose estimates on radon but does notaddress the potential for threshold effects. Published values are listed for effective dose frominhalation of radon and progeny using various dosimetric models for indoor, outdoor, home,
workplace and mines. The effective dose is dependent upon a number of factors but appears
to be age insensitive. Using the human respiratory tract model, values of effective dose per
WLM ranged from 10–20 mSv. For homes and mines, the effective dose was 13 mSv per
WLM, while it was 20 mSv per WLM for indoor workplaces, when the breathing rate wasadjusted to that of a standard worker. The Commission concluded that doses from radon andprogeny should be calculated using ICRP biokinetic and dosimetric models, which will allow
doses to be calculated to other organs besides the lung. Also, dose coefficients, equilibrium
factors and aerosol characteristics for domestic and occupational exposure conditions would
be provided subsequently.

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E.3. General architectural features of lung tissue

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(E26) Airflow passes through the upper respiratory tract (nasal passages, pharynx andlarynx) and then enters the lower respiratory tract. The airflow then enters a series of between
221 and 223 branches of the adult human airway (Fig. E.1.) covered by a pseudostratified
columnar epithelium in the larger branches (trachea and larger bronchi) of the conducting
system of the lung (Mercer et al., 1994; Weibel, 1963, 1997). As these branches in the human164DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
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lung become smaller, the epithelium becomes more cuboidal. Eventually, the smallest
branches merge with the alveolar epithelium where air exchange occurs (Fig. E.2.).

Fig. E.1. The adult human airway. (permission needed)

Fig. E.2. Illustrations depicting the major transitions in the human lung from large bronchi (central
airway) down to respiratory bronchioles connecting to the air exchange cells of the alveloli(peripheral airway). The cells lining the airways include ciliated and goblet cells in the central airway.
The basal cell is believed to have stem cell characteristics. The neuroendocrine cells may be involvedin small cell carcinoma. In the peripheral airways, there is a variety of types of Clara cells in addition
to cuboidal ciliated cells. In the alveoli, there are type I and type II pneumocytes. The stem cell(s) of
the peripheral airway are still somewhat controversial. (permission needed)(E27) The continuous layer of epithelial cells lining the conducting exchange portions of
the lung (termed the respiratory epithelium) plays a critical role in maintaining efficient
clearance of mucous, in defense processes, as well as for the passage of air (Knight andHolgate, 2003). There are about 30,000 terminal bronchioles (Lane et al., 2007) consisting ofClara domed-shaped cells, which have a protective and detoxifying function, and simple low
columnar ciliated cells. As the terminal bronchioles branch into respiratory bronchioles which
are contiguous with the septated saccular alveolar structures, the conducting portion of the165

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airways becomes the respiratory airways (where gas exchange occurs). In summary, at eachterminal bronchiole, several respiratory bronchioles emerge, and up to 11 alveolar ducts arise
from each respiratory bronchiole. These alveolar ducts each finally branch into about 5-6
alveolar sacs. To provide maximal surface areas for gas exchange to occur, there are about
300 million alveoli in the human lung giving a surface area of about 80 m2. The lung is also
unique among all internal organs by the way it is exposed directly and continuously to thesurrounding atmosphere in which hostile agents are often abundant. This could influence thesusceptibility of the lung parenchyma to inflammatory responses. Finally, the weight of thelungs is about 1.4 kg, and only the skin (2.7 kg), the liver (1.8 kg), and the blood (~7% of
total body weight) are heavier organs.
(E28) There are three cell types that are found within the alveolar spaces (Type I and TypeII pneumocytes, and alveolar macrophages). Type I pneumocytes are squamous cells thatcover over 95% of the alveolar wall, and lie in close apposition to the endothelial cells ofsmall capillaries where gas exchange occurs (Fig. E.2.). More cuboidal Type II pneumocytes
are interspersed among the Type I pneumocytes, and they secrete several surfactant proteinsthat reduce the surface tension within the alveolar sacs. In addition, the type II cells may havestem cell characteristics and help replenish damaged type I cells. The alveolar macrophages
do not attach to the alveolar wall and serve to engulf particles and infectious agents that havepenetrated the alveoli. They may also be important in mediating inflammatory responses.
(E29) While most of our knowledge of adult stem cells is based on experimental evidence
on the cells dedicated to the renewal of tissues such as the skin, gut and bone marrow, there ismounting evidence that organs such as the lung, liver, and pancreas, which turn over more
slowly, may use alternative strategies, including the self-renewal of more differentiated cells(Neuringer and Randell, 2004; Rawlins and Hogan, 2006). The response of these organs toradiation may also reveal the potential of differentiated cells to act as stem cells. The lungshows both slow turnover and rapid repair. New experimental approaches are needed to
identify putative lung stem cells and strategies of lung homeostasis and repair. The terms"stem cell" and "progenitor cell" are well defined in tissues with rapid turnover. Another termthat may be more appropriate for tissues with minimal turnover, except in cases of damage,
may be "reparative cells".
(E30) Several putative adult lung epithelial stem or reparative cells have been identified inmouse model systems. However, the in vivo capabilities of these putative stem cells tocontribute to different lineages, and their control mechanisms, remain unclear.

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Fig. E.3. One model of lung homeostasis (which is still controversial), includes endogenous cells fornormal lung turnover which is very slow, and both endogenous and exogenous cells that may berequired in the cases of acute injury to the lung. (permission needed)(E31) Many seminal studies with new isolation and characterisation techniques suggestthat stem cells exist in the adult lung. The picture that is emerging from mouse and rat models
is that different regions of the respiratory system, the trachea/large airways, and the distal
bronchioles and alveoli, use different populations of stem cells and strategies for maintenanceand repair (Fig. E.2.). Moreover, there is evidence that differentiated epithelial cell types areable to proliferate and trans-differentiate in response to some conditions. However, theprecise mechanisms involved in any of these processes are only beginning to be understood(Fig. E.3.).
E.3.1. Tissue turnover rate(E32) The steady-state turnover of epithelial cells in the lung and trachea is highly relevant
to investigations of endogenous adult stem cells, and the potential target cells of radiationassociated carcinogenesis. Early work indicated that the airway epithelium is a dynamic
tissue normally undergoing slow, but constant renewal. On the basis of studies inexperimental animals and limited studies in humans, the airway epithelium was believed
initially to renew every 30 to 50 days (Bowden, 1983). Upon injury, the airway epithelium
responds rapidly to reestablish an epithelial sheet with normal structure and function, withresident cells thought to be the source of the new cell population (Erjefalt 1995; Erjefalt andPersson, 1997). Although there may be some contribution from circulating stem/progenitor
cells, most evidence supports the concept that stem/progenitor cells distributed throughout the
airway epithelium are the source of the new epithelial cells, and that these stem/progenitor
reparative cells have the potential to differentiate into all of the cell types of the normal
epithelium (Rawlins and Hogan, 2006; Kim 2005). At present, there is no definitive
understanding of progenitor cell–progeny relationships in the airway epithelium. In addition,
there is not much evidence if “rare” lung epithelial stem/progenitor cells residing in specificniches (e.g. basal cells in the larger central airways and Clara or rarer c-kit+ cells in the
peripheral airways) proliferate, migrate, and differentiate in a highly orchestrated processsimilar to classical models of high turnover tissues.

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(E33) Recent studies have reexamined the average lifetime of different cell types in the
airways. Using a Cre/loxP genetic technology to label a random fraction of ciliated cells
throughout the airways of a cohort of mice, investigators followed the fate of these cells for
18 months (Rawlins and Hogan, 2008). The results demonstrated that ciliated airway
epithelial cells are a terminally differentiated population with a significant number of LRCs
still being present at the end of the experiment. The results demonstrated that the averagehalf-life of ciliated cells in the trachea is 6 months and approximately 17 months in theremaining compartments of the lung. Thus, in the absence of acute or chronic damage, theresidence time in the central and peripheral airway cells is much longer than previously
estimated (Rawlins and Hogan, 2008).
E.3.2. Age dependence: Lung development(E34) Based on morphology at different stages during embryonic and fetal development,
the following major stages of lung development have been described (Fig. E.4.). The stages
include the embryonic, pseudoglandular, canalicular, saccular and alveolar periods. The
embryonic stage (Fig. E.4., top) is when the lung bud emerges from the primitive foregut toproduce the trachea and major bronchial branches. The early growth of the emerging lungbuds is regulated by a series of growth factors and transcription factors including FGF10,
forkhead box A2 (FOXA2), TITF1, Sprouty, SHHH and TGFβ signalling. A pseudoglandular
stage between the embryonic and canalicular periods is when the lung resembles an
endocrine gland and branching continues to form bronchioles. At this stage, there is initiationof formation of ciliated and goblet cells. In the canalicular stage (Fig. E.4.), alveolar ducts
containing type I pneumocytes first appear. The saccular stage initiates the time when alveoli
ducts become sac-like structures which contain both type I and II pneumocytes. Finally,around birth and continuing after birth, the alveolar stage (Fig. E.4., bottom) involves the
final maturation of the alveolar sacs, including septation of the alveoli by the surrounding
mesenchyme and maturation of the vasculature surrounding the alveoli. Surfactant protein,which reduces surface tension, is secreted by the type II cells of the alveloli and Clara cells of
the smaller bronchioles.

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Fig. E.4. Development of the mammalian lung goes through specific stages. (permission
needed)E.3.3. Cellular features(E35) Basal cells in the lung central airways. Until recently, somatic stem cells in thepseudostratified mucociliary epithelium of the large airways remain largely unknown becauseof its cellular complexity, low baseline proliferation, and potential cellular plasticity ofdifferent cell types participating after injury. However, identification and isolation of specificcell populations from the trachea using advanced techniques are leading to the consensus thatat least one major class of lung stem cells, the basal cell, may represent at least one type oflung stem cells (Fig. E.5.).

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Fig. E.5. There is solid evidence that basal type cells in the central airways can differentiate into bothciliated and goblet cells. Much less is known about the neuroendocrine cells, but they are believed tobe capable of self-renewal and only into the additional neuroendocrine cell. It is more complex andcontroversial in the peripheral airways as to which cells have stem cell characteristics. There isevidence for a Clara or Clara-variant cell being important as well as type II cells dedifferentiating
upon acute injury. More recently, there is evidence that a rare c-KIT+ cell may exisit in niches in the
distal airway and be responsible for replacing most, if not all the cells of the peripheral airways.
(permission needed)(E36) Basal cells make up approximately 30% of the pseudostratified mucociliary
epithelium of the large airways of the lung (Rock et al., 2009). They are considered to be
undifferentiated, express p63, and express K5/K14 (Daniely et al., 2004; Evans et al., 2001;
Schoch et al., 2004). In the rodent, basal cells are confined to the trachea, where they areinterspersed among the ciliated, secretory (goblet), and neuroendocrine cells (Fig. E.5.). In
contrast, in the human lung, basal cells may be present throughout the airways, including
small bronchioles (Boers et al., 1999; Evans et al., 2001; Nakajima et al., 1998).
(E37) While cell turnover in the adult trachea is normally very low (Kauffman, 1980),
epithelial injury elicits rapid proliferation of surviving cells, except ciliated cells (Rawlins etal., 2007), and the tissue is soon repaired. Several lines of evidence suggest that tracheal basal
cells function as stem cells for this repair. First, lineage tracing of K14-CreER–expressing
cells after naphthalene injury, which depletes secretory goblet cells, demonstrates that somebasal cells can both self-renew and give rise to ciliated and goblet cells (Hong et al., 2004).
Second, a subset of basal cells retain BrdU label in the long-term after epithelial damage by
SO2 inhalation (Borthwick et al., 2001). Third, basal cells isolated on the basis of highexpression of a K5-GFP transgene have a greater capacity to proliferate and give rise tolarger colonies in vitro compared to K5-GFP− cells (Schoch et al., 2004). Fourth, fractionated
rat tracheal basal cells can restore the entire epithelium of a denuded trachea in a xenograft170

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model (Randell et al., 1991). Finally, isolated basal cells of the mouse trachea function asprogenitor cells both during postnatal growth, in the adult at steady state, and in a repair
model. Using a clonal assay, basal cells of both mouse and human airways can self-renew
and differentiate in the absence of stroma or columnar epithelial cells (Rock et al., 2009).
Basal cells do not produce neuroendocrine cells, and these appear to be another type of stem
cell in the lung, but neuroendocrine cells appear to give rise only to other neuroendocrine
cells (Fig. E.5.).
(E38) BASCs, Clara, Clara variant, and facultative stem cells of the peripheral airways.
The existence of distinct proximal (tracheobronchial, large bronchiolar) and distal (smaller
bronchiolar and terminal bronchiolar) stem cells hierarchies suggests that discrete
regenerative units maintain the conducting airway epithelium. Characteristics of the proximaland distal epithelial compartments are potentially a reflection of distinctions in the propertiesof region-specific stem cells, as well as the influence of the niche/microenvironment on theactivity of these cells. While in the trachea and large bronchi, the basal cell is considered tobe the main stem cell type, in the more distal airways (small bronchi and bronchioles), theepithelium changes from columnar to cuboidal and Clara cells predominate over ciliated cells(Fig. E.5.). Importantly, there are no basal cells in mouse or rat peripheral lung, so that they
are unlikely to be involved in turnover and repair (Pack et al., 1981; Randell et al., 1991) of
the small bronchioles or alveoli. However, in the human lung, some type of basal cells may
persist in the distal airways, although this remains uncertain.
(E39) The most distal region of the lung is organised into a complex system of alveoli,
where there are two types of epithelial cells. Type I cells (also called type I pneumocytes),
provide the thin-walled gas exchange surface. Type II pneumocytes contain numerous
secretory vesicles (lamellar bodies) filled with surfactant material, including surfactant
protein C. There is evidence that type II pneumocytes are another peripheral stem cell of thelung, but that type II cells give rise only to other type II cells. Also, when there is acutedamage, type II cells are capable of replicating and differentiating into type I cells (Fig. E.5.).
(E40) The transitional region between the terminal bronchioles and the alveoli is known as
the bronchioalveolar duct junction (BADJ). There is a variety of cell types at this junction:
one such cell type expresses SCGB1a1, also known as Clara cell 10-kDa protein (CC10) or
Clara cell secretor protein (CCSP), and is often used as a marker for Clara and Clara variant
cells. There have been many studies of SCGB1a1 expression in relation to cell lineages in
mice, but in some instances, the definition of the Clara cell has become confusing, and there
may be more than one type of Clara cell (Fig. E.5.). If Clara cells are morphologicallydefined as domed cuboidal, nonciliated cells of the simple bronchiolar epithelium as
originally described, then SCGB1a1-expressing secretory cells in the pseudostratified
portions of the airway are distinct from bronchiolar Clara cells. However, Clara cells arecommonly defined based on SCGB1a1 expression. This suggests that Clara cells likely
represent a spectrum of cells, present in different airway levels, with different stem cellpotentials. A generic term for this spectrum of cells is a facultative stem cell. This term refers
to a differentiated cell that is normally quiescent but responds to injury by dividing and selfrenewing, and giving rise to progeny that differentiate into one or more cell types.
(E41) After injury by oxidant gases that damage ciliated cells, surviving bronchiolar Claracells proliferate to restore the bronchiolar epithelium (Evans et al., 1986); but this observation,in itself, does not provide evidence for a stem cell hierarchy within the Clara cell population.
When naphthalene is administered to mice, almost all Clara cells are killed due to selective
metabolic activation of the toxin. Ciliated cells shed their cilia and cover the denuded
bronchiolar basement membrane, and the few surviving Clara cells (some term these Claravariant cells) then proliferate (Van Winkle et al., 1995). The naphthalene-resistant progenitor
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cells represent a subset of Clara cells that are label retaining thus suggesting that this may
constitute the actual distal lung stem cell (Hong et al., 2001). After naphthalene injury,
pulmonary neuroendocrine cells also proliferate (Stripp et al., 1995), but they are a distinct
lineage system not requiring nor generating Clara cells (Reynolds et al., 2000). These aresimilar to the neuroendocrine cells found in the larger airways. An epithelial stem cell niche
has been identified in the zone where airways terminate and form alveoli (Giangreco et al.,
2002; Hong et al., 2001). Specific cells in the bronchiolar alveolar junction, co-express
SCGBa1a, the type II cell marker surfactant protein C, Sca-1 (Kim et al., 2005), and are
negative for CD45 and CD31.
(E42) The putative mouse BASCc proliferate in response to naphthalene or bleomycin
injury, and demonstrate a high clonal growth capacity and differentiation potential to form
both Clara cells and distal lung epithelium composed of cells expressing type I or type II cellmarkers (Kim et al., 2005). There are only limited data suggesting that BASCs can form typeI or II cells, and these are based on cell markers. There are no direct in vivo data
demonstrating that isolated BASCs can form alveolar cell types, and it is entirely possiblethat type II cells may dedifferentiate upon damage and resemble BASCs. In mouse models oflung cancer, BASCs expand when an active K-ras oncogene is induced to generate ADCs
(Kim et al., 2005). Thus, Clara, Clara variant, BASCs, or facultative stem cells exhibit transit
stem cell characteristics after injury, and may contribute to lung cancer. This is supported by
Xu et al. (2012), where, using a Cre-knock-in approach to express a mutant K-Ras in CC10+
epithelial cells and surfactant protein C-positive type II alveolar cells in an adult mouse, type
II cells, Clara cells of the terminal bronchiole and BASCs were identified as the cells oforigin for K-Ras-induced lung hyperplasia. Interestingly, only type II cells progressed to
ADC.
(E43) In summary, an area of much uncertainty is if Clara, Clara-variant, or BASC cells
can actually give rise to type I or II cells. However, the regeneration of type I and II cells
after injury using a transgenic mouse model where Scgb1a1-expressing cells and theirprogeny can be labelled with the enhanced GFP (EGFP) was described (Zheng et al., 2012).
They argued that these EGFP-expressing progenitor cells are most likely Clara cells and it isthese cells that respond to injury of the alveolar epithelia after bleomycin exposure. Given thesimilarity of the lung response between bleomycin and radiation injury, it is conceivable thatthe same model would be appropriate to test the radiation response.
(E44) It is also important to determine if similar multipotent cells exist in the humanbronchiolar–alveolar duct junction zone, as it does in the mouse. More detailed in vivo
molecular and cellular characterisation of BASCs, other putative lung stem and progenitor
cells, and differentiated cells, is needed to determine the lineage relationships in the adulthuman lung. The studies conducted so far highlight the existence of region-specific stem cells
in the conducting airways and the complexity of analysing the differentiation potential ofstem cells at different sites. While most knowledge about peripheral lung stem cells has beenobtained using mice, a major deficiency is our understanding of these processes in the humanlung. Recently, but controversially, there is evidence for the existence of a human lung stem
cell expressing c-KIT that may be able to replace all the cells in the distal lung (Kajstura et al.,2011). This cell type is proposed to reside in rare niches in the distal lung and also to becapable of regenerating vascular endothelial cells after lung injury. These c-KIT+ cells also
express the same transcription factors that are present in ES cells (Nanog, Oct 3/4, KLF4 andSox2), suggesting that they may actually be exogenous cells from the bone marrow.
(E45) Human lung diseases such as cystic fibrosis or chronic obstructive pulmonary
disease, idiopathic pulmonary fibrosis, as well as the most common forms of lung cancer in
the US, may involve bronchiolar and alveolar cell defects. It is likely that the delicate balance
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of stem, progenitor, and differentiated cell functions in the lung is critically affected inpatients with these devastating diseases. Thus, the discovery and a complete understanding of
putative lung stem cells will lay the foundation for new inroads to understanding lung biology
related to lung disease.
(E46) Lung stem cell summary. Adult stem cells are still ill defined in the human lung, andthe mechanisms that control their proliferation and differentiation are almost completely
unknown. Nevertheless, the possibility that lung disorders may one day be treated bymanipulating endogenous lung stem cells, or with exogenously applied stem cells, is thefocus of much current research effort. However, if this approach is to be successful, it isnecessary to understand the normal behaviour of endogenous lung stem cells, and how they
are controlled by their environment.
(E47) Adult marrow-derived cells in lung biology and disease. There is mounting interestin the role of bone marrow-derived progenitor cells in the repair of massive and chronic lungdamage. While there are a number of current controversies regarding assessments of lungchimerism by adult bone marrow-derived cells, at the present time, a consensus on thefunctional role of adult marrow-derived cells in lung repair and remodelling after injury has
not been clarified (Fig. E.3.). It is well established that adult bone marrow cells serve asprecursors for HSCs. However, a substantial number of studies report that cell populations
derived from bone marrow of adults including HSCs, stromal-derived MSCs and circulating
fibrocytes, can localise to and acquire phenotypic and functional markers of mature tissues
including lung (Pereira et al., 1995; Suratt et al., 2003) (Fig. E.2.). Whether the bonemarrow–derived cells are truly "stem" cells has not been rigorously demonstrated, and mostof these studies are controversial. In some cases, the marrow-derived cells may fuse withexisting organ-specific cells rather than trans-differentiating into mature organ-specific cells.
For example, in humans, lung specimens from some clinical bone marrow transplant
recipients demonstrate chimerism for both epithelial and endothelial cells (Mattsson et al.,2004). Similarly, lung specimens from some lung transplant patients demonstrate chimerismof lung epithelium (Kleeberger et al., 2003; Spencer et al., 2005). However, chimerism orlung engraftment with adult marrow–derived cells has not been found in all studies.
Irrespectively, even fusion of normal adult marrow–derived cells with irradiateddifferentiated adult lung tissue may influence our current models of lung cancer risk
assessment after ionising radiation.
(E48) In summary, using more recent sophisticated techniques, the current consensus isthat local cells within the lung are primarily responsible for maintaining or reconstituting thelung epithelium, and that bone-marrow derived cells contribute few, if any, cells directly tothe structure of the airway or alveolar epithelium under normal homeostatic conditions.
(E49) Molecular characterisation of lung cancer. As briefly described above, lung cancer
develops in a series of steps extending over years. Conceptually, this is divided into 3 phases:
initiation, which is the accumulation of genetic and epigenetic changes; promotion, where
there is a selective growth of cells with these changes over normal cells; and progression,
which leads to the development of invasive cancer and the metastatic phenotype. Lungcancers are of three main types: SCLC; non-small cell lung cancer (NSCLC); and ADC.
NSCLC accounts for 85% of all lung cancers. Lung cancer is principally caused by tobaccosmoke: 98% of all SCLC patients and 85% of all NSCLC patients smoke. And, while thenumbers of lung cancers is decreasing in the US and the UK, in China 2/3 of all adult males
smoke and they represent 1/3 of all smokers worldwide. Interestingly, some 20% of ADCs
occur in non- or never-smokers, arise predominantly in the peripheral lung, and thedevelopment of these tumours is unique from those lung cancers seen in smokers or former
smokers (Sun et al., 2007). Epidemiological studies have also identified a major lung cancer
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susceptibility locus at 6q23-25 that is associated with a 2.5-fold increased risk (Amos et al.,
1999; Bailey-Wilson et al., 2004).
(E50) NSCLC, SCLC and ADCs arise from different compartments of the lung, and arethought to arise from one of two putative stem cells that are found in different locations of thelung and which are responsible for specific differentiated cell types. The basal bronchial cellfound in the central region of the bronchial airways can differentiate into ciliated or mucosal
cells and can give rise to ADC, NSCLC and SCLC. The BASC, or Clara cell, gives rise to theterminal respiratory unit of bronchioles and alveoli which can give rise to peripheral ADCs.
(E51) There have been extensive molecular and genetic studies of lung cancer (Ding et al.,2008; Sekido et al., 2003; Thomas et al., 2007; Weir et al., 2007) as recent examples which
describe genetic and epigenetic changes associated with the development of lung cancer.
Indeed, detailed microdissection and molecular analyses of the bronchial epithelium frompeople with and without lung cancer have described histologically normal bronchial
epithelium that contains clones of cells with genetic changes seen along the pathway to lungcancer, while bronchial brushes and sputum of people exposed to cigarette smoke without
lung cancer contain DNA methylation (epigenetic) changes (Wistuba et al., 2000, 1997;
Zochbauer-Muller et al., 2003). The genetic alterations that occur in the development of lungcancer are substantial. Sekido et al. (2003) describe more than 20 alterations, genetic andepigenetic, in overt lung cancers. These alterations can and should be categorised by the
biological consequence of the signalling pathways altered.
(E52) Tumour suppressor genes. p53, the most frequently mutated gene in lung cancers
impinges upon several signalling pathways (Olivier et al., 2009). Mutations within the DNAbinding region of p53 can result in inactivation, or gain of function mutations can result in
mdm2 binding and subsequent p53-mediated proteolysis. There is a distinct mutational
spectrum described for smoking. Upstream regulators of p53 include mdm2 and p14. Bothloss and gain of function alterations in either protein result in the disregulation of p53. Lossof p14 is a frequent event in SCLC, large cell neuroendocrine cancer (LCNEC) but less so in
ADC (Brambilla et al., 1998). Altered p53 signalling has adverse consequences for cell cycle
checkpoint control, regulation of either pro- or anti-apoptotic proteins via mitochondrial
apoptosis, or death receptor signalling via the FAS/tumour necrosis factor-related apoptosisinducing ligand (TRAIL) pathway, and DNA repair.
(E53) A lack of retinobalstoma protein Rb is also common to some lung cancers. Rb
regulates G0/G1 transition via cyclin D, and is the most frequent factor in loss of cell cycle
control in SCLC. Interestingly, hyperphosphorylation of Rb is more common in NSCLC. The
mutation status of Rb in ADC also reflects a loss of RB function through either mutation,
LOH, or hyperphosphorylation (Ding et al., 2008; Weir et al., 2007). Hyperphosphorylationin NSCLC is via loss of p16 or overexpression of Cyclin D1 (40-60% of NSCLC), and p16
activity can be suppressed via methylation, deletion or mutation (Brambilla et al., 1999; Weiret al., 2007). These are early events associated with dysplasia. Cyclins D and E are frequently
found amplified in ADC (Weir et al., 2007), and overexpression is an early event in bronchialpre-invasive lesions (Jeanmart et al., 2003).
(E54) Oncogene addiction. Oncogene addiction refers to the addiction of a cell to theabnormal function of oncogenic proteins. These oncogenic proteins generally drive cells
toward proliferation and subsequent malignancy. In lung cancer, it is members of the RTK
family that likely drive oncogenic processes because of their position at the pinnacle of anumber of signalling pathways that control cell cycle and proliferation, angiogenesis, evasionof apoptosis, and DNA repair. In particular, the EGFR, one of 4 RTKs that homo- or
heterodimerise, allow for a diverse set of cytoplasmic signalling events. These signalling
events include activation of the phosphotidylitositol 3-kinase (PI3K) pathway, signal
174

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transducers and activators of transcription (STAT) signalling and the RAS pathway. EGFR isoften found amplified in cancer; however in NSCLC, EGFR is often mutated as well as
amplified (Eberhard et al., 2005; Hirsch et al., 2003; Shigematsu and Gazdar, 2006). For
ADCs, geographical location, never-smoking status and female sex are independently
associated with EGFR mutation, and EGFR overexpression is associated with poor prognosis(Nicholson et al., 2001). Mutations in EGFR target the tyrosine kinase domain, and include
mutations, deletions, insertions and activating point mutations. A point mutation resulting inan arginine for leucine substution at amino acid 858 (L858R) and a deletion in exon 19
account for approximately 85% of all mutations. EGFR has become a significant biologicaltarget for targeted therapy.
(E55) The PI3/v-akt murine thymoma viral oncogene homolog (AKT) pathways, which are
regulated by ERBB members, lead to enhanced cell survival and proliferation (Sharma et al.,2007). PI3K catalytic subunit p110α (PI3KCA), along with KRAS is a gene found mutated inmany cancers; however, amplification of the 3q25-27 region, which contains PI3KCA, often
occurs in NSCLC and especially in SCC (Garnis et al., 2006). PI3KCA is one of the few
known genes that is found more frequently disregulated in SCC than ADC.
(E56) Similar to EGFR, the extent of mutations in KRAS (10-20% in NSCLC) is
associated with geographical location. However, KRAS mutation is rare in Asians. KRAS
mutations are strongly associated with ADC and appear more in males and smokers. KRAS
mutations appear to target smokers (Gazdar et al., 2004). Inactivating mutations within theliver kinase B1 (LKB1) gene are often associated with KRAS tumours. These inactivating
mutations are associated with the loss of barriers to initiation and altered cell polarity andmetastasis (Ji et al., 2007). LKB1 mutations are frequently seen in ADC, although they areuncommon in most tumours (Herbst et al., 2008; Matsumoto et al., 2007).
(E57) Other oncogenic addictions may include TITF1, which codes for the TTF1 protein,
and which is a lineage-specific marker for peripheral ADCs. These tumours of the terminal
respiratory unit usually express TTF1, and when deprived, will undergo growth arrest or die
by apoptosis (Tanaka et al., 2007). Lastly, in regard to oncogenic addiction, there is
amplification of the MYC family. The MYC genes control cell growth and apoptosis, andthey are often amplified in lung cancers. c-MYC is more often associated with NSCLC while
L-MYC is predominantly associated with SCLC. In either cancer, enhanced MYC expressionis also seen as a result of transcriptional upregulation without gene amplification (Gazzeri etal., 1994; Gazzeri et al., 1990).
(E58) Evasion of cell death. Mitochondrial apoptosis is regulated by the interaction of the
BAX and BCL-2 proteins. The anti-apoptotic protein BCL-2 blocks the activity of the proapoptotic protein BAX through dimerisation (Danial, 2007). BCL-2 is overexpressed in mostSCLC, and to a limited extent, in NSCLC (Brambilla et al., 1996; Gazzeri et al., 1998). BAX
on the other hand is downregulated in SCLC, and upregulated in NSCLC. The result is
dramatically altered BAX/BCL2 ratios that favour cell survival in NSCLCs, and this is seen
early in the generation of dysplasia (Brambilla et al., 1998). Impinging upon the apoptotic
pathway via caspase-8 activation is the Fas signalling pathway, which is activated when Fas
ligand (FasL) binds the death receptor Fas. Both Fas and FasL are downregulated in the
majority of NSCLCs. This is in contrast to SCLC where the Fas receptor is downregulated or
not expressed at all, yet FasL is highly expressed. This disrupts the apoptotic pathway
ordinarily regulated by the Fas receptor.
(E59) Normally, the shortening of telomeres, the repetitive sequences at the end ofchromosomes that stabilise chromosome integrity and chromosome length, shortens after
each cell division. This limits the lifetime of cells through the activation of apoptosis orcellular senescence. Because cells see short telomeres as DNA damage, the inactivation of175

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p53 signalling limits the activation of DNA repair and cell cycle checkpoint pathways at theearly stages of the carcinogenic process (Bartkova et al., 2005; Gorgoulis et al., 2005;
Nuciforo et al., 2007). Without p53 or Rb checkpoint controls, telomerase activity is
upregulated which, through selection, results in production of immortalised cells that are
genomically unstable. Human telomerase reverse transcriptase (hTERT), which maintains
telomere length, is overexpressed in alveolar hyperplasia (75%) and in some bronchoalveolar carcinomas (Lantuejoul et al., 2004), and is seen in 80% of NSCLC and nearly all
SCLCs (Hiyama et al., 1995b; Lantuejoul et al., 2004).
(E60) Molecular alterations. Molecular epidemiological studies have now described
different molecular alterations in never-smokers when compared to lung cancer in smokers,
suggesting that these cancers arise by different mechanisms. For example, mutations in both
KRAS and EGFR are associated with ADCs (Chiosea et al., 2007; Johnson et al., 2001; Politi
et al., 2006). However, mutated KRAS is found almost exclusively in lung cancers fromsmokers, while EGFR mutations are more frequent in never-smokers. In fact, EGFR
mutations were the first specific genetic mutation associated with never-smokers (Kosaka etal., 2004; Pham et al., 2006). p53 mutations, while occurring in both smokers and neversmokers, occur less frequently in tumours from non-smokers. Furthermore, the mutational
spectrum for p53 is unique to smoking status (Denissenko et al., 1997; Le Calvez et al., 2005;
Vahakangas et al., 2001). More broadly, a number of studies examining gene expression haveshown the expression profiles to be very different from one another (Gorgoulis et al., 2005;
Lam et al., 2006; Takeuchi et al., 2006), including high levels of expression of AKT and p27(Dutu et al., 2005). Methylation patterns in lung cancers identified in never-smokers versus
smokers may also be distinct (Marchetti et al., 1998).
(E61) Given that over 50% of lung cancer in females are seen in never-smokers, while thesame can be said for only 15% of male never smokers, the likelihood of sex-specifichormones as a risk factor for lung cancer is possible. Estrogen metabolism, for example, may
result in mutagenic DNA adducts (Cavalieri and Rogan, 2006; Yager and Liehr, 1996). Other
studies have looked at the role of estrogen as a proliferative agent in NSCLCs, as well as therole of ERα and ERβ. ER, for instance, is seen more often in tumours from never-smokers
and more frequently in tumours from females rather than male never-smokers (Wu et al.,2005). Expression of ER is also considered to be a negative therapeutic prognosis factor in
NSCLCs (Kawai et al., 2005). Mechanistically, however, it is the interaction of the EGFR
and ER pathways that is most compelling. When estrogen is stimulated in lung cancer celllines, EGFR ligand is rapidly released, which then activates the EGFR/MAPK1 growth
pathways (Stabile et al., 2005). Indeed, EGFR protein expression is downregulated in thepresence of estrogen and upregulated in the presence of anti-estrogens. Furthermore, in amouse model of lung cancer that employed a conditional knockout of K-ras and deletion of
p53, female mice had twice the number of tumours, larger tumours, and tumours of a higher
grade, compared to male counterparts, a finding that was eliminated in ovariectomised
females (Hammoud et al., 2008).
(E62) Radon was the first environmental cause of lung cancer identified. As early as the
1920’s, lung cancer was attributed to radon exposures in underground miners. The risk forlung cancer in underground miners classified as never-smokers as a result of radon exposure
has been shown to be elevated in several studies (Archer et al., 1973; Gilliland et al., 2000;
Roscoe et al., 1995; Roscoe et al., 1989; Samet et al., 1984). The determination of cellularand molecular features of radiation-induced cancers developed in parallel to thedetermination of the molecular phenotype of lung cancer in general. Indeed, risk models forradiation-induced lung cancers are now beginning to incorporate such biological markers oflung cancer.
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(E63) One of the first studies to examine the molecular phenotype of radiation exposedlung cells was that of Vahakangas et al. (1992), who described the mutational spectrum of the
p53 and Kras genes in underground uranium miners. Interestingly, they observed a different
mutational spectrum for lung cancer for radon exposure compared to that of tobacco smokers.
They identified no kras mutations in exons 12-13. Although 9 mutations were seen withinexons 5-9 of the p53 gene, none of these mutations were in the hotspot codons described forlung cancer. This study was followed up by several other studies in different populations ofminers. In 1994, Taylor et al. (1994) described a single mutation at codon 249 of the p53
gene as a mutation hotspot in radon-associated lung cancer, including non-smokers. This
hotspot was not found in either of three comparable datasets (Bartsch et al., 1995; Tierney etal., 1996; Yang et al., 2000) nor in a study of lung tumours associated with domestic radon
exposure (Lo et al., 1995). In a recent analysis of p53 mutational status, Ruano-Ravina et al.
(2009) examined the data from 8 studies that included 578 individuals, and concluded that theresults do not support a radon-associated mutational hotspot for the p53 gene.
(E64) A number of animal systems including mouse, rat, dog, and baboon have been used
to determine biological data for lung cancer induction via inhalation of radioactive elements
such as radon or plutonium (Bair WJ, 1989; Bruenger et al., 1991; Guilmette et al., 1987;
Lundgren et al., 1995; Morgan, 1989; Oghiso et al., 1994; Sanders et al., 1993; Simmons andRichards, 1984; Yuile et al., 1970) or irradiation via external sources. The role of KRAS or
p53 mutations in radiation-induced lung carcinogenesis was examined in γ-irradiated F344/N
rats (Belinsky et al., 1996) and in plutonium exposed rats (Stegelmeier et al., 1991). Uniqueto these studies was the identification of K-ras mutations in 33% of tumours after 239PuO2
exposure, yet only a single K-ras mutation was identified in 35 x-ray induced tumours (18squamous and 17 adeno). This suggests that there is not a specific signature nor a major rolefor p53 or K-ras mutations in lung carcinogenesis after low-LET radiation, but that there may
be for high LET radiation. As suggested by Tierney et al. (1996), high-LET radiation-induced
cancers may be through species-specific molecular mechanisms, as their studies of p53 andK-ras mutations in canines differed from those of Stegelmeier et al. (1991), who employedrats as their model system, but concurred with those of Vahakangas (Vahakangas et al., 1992)
for K-ras mutations. p53 mutations, on the other hand, were one-third of those seen in lungcancers in uranium miners (Taylor et al., 1994; Vahakangas et al., 1992), and in line with thefrequency seen in rat lung tumours (Kelly et al., 1995; Leach et al., 1993), again suggestingthat the p53 abnormalities associated with radiation-induced lung cancers in animals andhumans may be different.
(E65) Epigenetic events. Regulation of gene expression without changing DNA sequenceis referred to as epigenetic regulation. This can occur through methylation of CpG islands
within genes and their promoter regions, which silences the gene, or through gene regulation
by miRNA. In addition, loss of methylation in non-coding regions is common in tumours,
and is considered to lead to genomic instability. There are many genes that have beensilenced in lung cancers (Hanahan and Weinberg, 2000; Shames et al., 2006), including
retinoic acid receptor β (RARB), p16, O6-methylguanine-DNA methyltransferase (MGMT),
tissue inhibitor of metalloproteinase 3 (TIMP3) and Ras association domain family member
1A (RASSF1A) as examples (Zochbauer-Muller et al., 2001), and this process of genesilencing begins early in the pathogenesis of lung cancer (Zochbauer-Muller et al., 2003).
Epigenomic analysis has shown that alterations of histone proteins are linked with DNA
methylation patterns and is likely causal for lung cancer (Esteller, 2007).
(E66) miRNA, non-coding small RNAs, regulate gene expression by controlling theactivity of specific mRNA via precise base pairing interactions (Boyd, 2008; Cowland et al.,2007), and miRNA expression is now known to be disregulated in lung and other cancers
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(Nana-Sinkam and Geraci, 2006; Ramdas et al., 2009; Takamizawa et al., 2004; Yanaihara etal., 2006). The targets for most miRNAs are as yet unknown; however, they can clearly act ina manner analogous to tumour suppressors or oncogenes through their targeted regulation ofmRNA levels, or through translational silencing. Interestingly, they are often found at DNA
fragile sites and in regions of amplification or deletion. Six miRNAs are unique to ADC. Let7 is decreased in lung cancer and is known to regulate KRAS expression (Eder and Scherr,2005), which is also mutated in many ADCs. Let-7 also regulates expression of cell cyclerelated genes, cellular division and genes in the DNA repair pathways. miRNA 34 (Mir-34) is
now linked to the p53 pathway including cell cycle arrest and apoptosis (He et al., 2007), andthe deletion of miR-34 is seen often in lung cancer (Bommer et al., 2007; Calin et al., 2004).
Other miRNAs impact cell cycle through myc activity (Hayashita et al., 2005), and the
DICER protein, which is a miRNA-processing enzyme, is decreased in pre-invasive lesions
in ADC hyperplasia (Chiosea et al., 2007).
(E67) Epigenetic events associated with radiation exposure. As described above, altered
DNA methylation patterns play a significant role in the lung carcinogenic process. Lyon et al.
(2007) have described the methylation of the promoters of specific genes in ADCs of Mayakworkers. Comparisons for a 4-gene panel in workers versus non-workers identified
significant increases in methylation frequency for p16 and GATA-binding protein 5(GATA5), as well as a dose response for plutonium exposure and the extent of gene
methylation (Belinsky et al., 2004; Lyon et al., 2007). Furthermore, in addition to the increase
in methylation, the multiplicity of gene inactivation was enhanced with increasing dose.
Comparing the ADCs of γ-ray exposed versus plutonium-exposed individuals did not reveal a
difference in the extent of multiplicity, suggesting that LET played no role.
(E68) Leng et al. (2008) argued that DSB repair, as measured by chromosome/chromatid
rejoining capacity, influenced the extent to which genes were methylated. Using the Lovelace
Smokers Cohort, single nucleotide polymorphisms in the DSB repair pathway [MRE11,
checkpoint kinase 2 (CHEK2), x-ray repair cross-complementing group 3 (XRCC3), DNAPKcs, and nibrin (NBN)] were scored. As the number of single nucleotide polymorphisms(SNPs) within these genes increased, the risk for gene methyation also increased. When thenumber of SNPs rose to seven, there was a 14.5-fold risk for a high methylation index. Also,
although this cohort has no association with radiation exposure, these data support the notionthat there are individuals who will carry a greater risk for promoter methylation, and perhaps
carcinogenic risk, after radiation exposure based upon their intrinsic radiosensitivity via theircapacity to repair DNA damage.
(E69) Genetic factors. Even though lung cancer is considered a disease related toenvironmental factors, some 80-90% of smokers will not get lung cancer. This suggests thatfor the 10-20% that develop lung cancer, genetic factors may play an important role in thesusceptibility to the environmental factors that cause lung cancer in never-smokers.
Epidemiological studies identifying familial clustering have shown an increased risk for lungcancer in never-smokers (Matakidou et al., 2005), racial differences (Cote et al., 2005), and amajor susceptibility locus for lung cancer at 6q23-25 (Bailey-Wilson et al., 2004). Thissuggests that genetic factors play an important role in lung cancer incidence.

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E.4. Radiosensitivity: Cells involved in lung repair after damage, and radiation as alung carcinogen

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(E70) After injury by bleomycin, naphthalene, sulphur dioxide and irradiation, epithelial
repair is rapid with both dedifferentiation and migration of cells to cover defects, as
determined by metabolic pulse-labelling studies. This not only indicates a substantial

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plasticity of cells in the airways, but also that there is a high priority to repair such epithelial
injuries. Even though there have been impressive recent research advances, a major area of
uncertainty is the number, sensitivity, location and renewal characteristics of putative lungendogenous stem cells. In the respiratory tract, the target cells for radiation-associatedcarcinogenesis are considered to be in the basal cells in the trachea and larger bronchi of thecentral lung (Hong et al., 2004; Rawlins et al., 2007; Rock et al., 2009; Schoch et al., 2004),
and in the Clara variant and type II alveolar cells of the peripheral lung (Giangreco et al.,
2002; Kim et al., 2005). While it is commonly believed that the target cell of radiationassociated carcinogenesis is the stem or progenitor cell of various tissues, very little is known
about radiosensitivity of specific stem cell populations in the lung. In addition, while in manytissues, the stem cell niche may be protected from atmospheric oxygen and potential ROS,
the lung is chronically exposed to oxygen.
(E71) The question arises as to the target cell for carcinogenesis. As described by
Greenberger and Epperley (2009), there are two distinct possibilities. The first possibility isthat radiation-induced cancers are the product of malignant transformation of stem cells
found within the irradiated field at the time of irradiation. The second possibility is that
MSCs have migrated into the irradiated site and that it is the proliferation of these cells, in a
microenvironment of increased free radical production in association with the onset of
fibrosis, that is responsible for carcinogenesis. The appearance of donor origin cell markers ina number of studies of relapsed leukaemia in marrow transplant recipients (Katz et al., 1993;
Thomas et al., 1972), and the donor bone marrow origin of gastric cancer in chronicallyinflamed Helicobactor pylori-infected mice (Houghton et al., 2004) suggest at least thepotential for the second mechanism. While there is evidence that bone marrow-derived cells
contribute to both tissue repair and late fibrosis of irradiated epithelial tissues (Epperly et al.,
1999; Greenberger and Epperly, 2009), the evidence for mesenchymal stem celldifferentiation into lung epithelial cells is controversial. As described earlier, marrow-derived
cells may fuse with existing organ-specific cells rather than trans-differentiating into matureorgan-specific cells. While this would limit the likelihood of the second mechanism inradiation-induced carcinogenesis, fusion of normal adult marrow–derived cells with
irradiated differentiated adult lung tissue may influence our current models of lung cancer
risk assessment after ionising radiation exposure.
(E72) In summary, there is a large knowledge gap regarding the radioresponse ofendogenous lung stem cells: the extent to which stem cells are radiosensitive is unclear (Baoet al., 2006; McCord et al., 2009; Woodward et al., 2007); and any role for MSCs in lungcarcinogenesis is controversial. Other questions concern the role of the microenvironment. If
the MSC hypothesis is true, then the microenvironment is critical. Is the target cell dependent
upon radiation delivery, e.g. inhalation vs external irradiation? After the initiating event(s),what molecular alterations are associated with progression?
(E73) In addition, it is increasingly clear that the molecular phenotype of radiation-induced
lung cancer may be species specific, given the differences in radiation-induced molecular
phenotypes between human, dog, and rodent. In humans, K-RAS mutations, commonly
associated with lung cancer in smokers, appear not to be linked to radiation-induced lungcancers when examined in radiation transformed lung epithelial cell lines or in humans after
domestic radon exposure, which is similar to K-RAS status in lung cancers in never-smokers.
Epigenetic changes, however, are seen in both smoking-induced cancers and radiationinduced cancers. Indeed, there may be a radiation dose response for gene promoter
methylation, although LET appears not to play a role (Goetz et al., 2011). Almost completely
ignored is the possibility that there is an interplay between hormonal regulation (estrogen)and radiation exposure in the increased risk for lung cancer seen in women, particularly non179

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smoking women. Is estrogen exposure a promotional event for initiated stem/progenitor
cells? Finally, while we may be gaining insight into radiation-induced lung cancer bycharacterising the molecular phenotype, the link between stem cell/progenitor cells and overt
lung cancer after radiation exposure has yet to be bridged.
E.4.1. Single cell responses: Endogenous lung tissue stem cells(E74) While multipotent long-lived cells have been identified in distinct regions of thelung architecture, the identification of undifferentiated human lung stem cells has remained
elusive. Only recently, such a cell type within the lung has been identified (Kajstura et al.,2011), based upon the expression of c-kit and negative for a host of markers of cell type. The
consequences of radiation exposure in these cells have not yet been studied.
(E75) However, non-oncogenically immortalised and oncogenically immortalised
bronchial epithelial cells have been used to elucidate molecular and cytological responses toradiation associated with the oncogenic process. Based upon the incidence of breast cancer in
A-bomb survivors, the carcinogenic risk for epithelial cells has been estimated at 10-12/Gy percell (Hei et al., 1996; Hei et al., 1998; Zhou et al., 2004). Using an oncogenicallyimmortalised [human papilloma virus (HPV)-immortalised] normal human bronchial
epithelial cell system referred to as BEP2D cells, Hei et al. (1996, 2001) described malignant
transformation of BEP2D cells after 4He- and 56Fe particle irradiation as measured by cellulartransformation, e.g., growth in soft agar, to tumour production in immune-compromised mice
after injection of transformed cells. Interestingly, no KRAS mutations were identified in the4
He-particle or 56Fe-irradiated cells and subsequent tumours. However, Betaig-h3 was
identified as causally linked to tumour induction in BEP2D cells after 56Fe irradiation (Zhouet al., 2004). Betaig-h3 expression is regulated by TGFβ1 signalling and when reintroduced
into 3 tumourigenic cell lines, tumourigenicity was markedly reduced. In addition, mutated
p53 was seen in BEP2D tumours where no p53 was seen in the unirradiated cell line. Because
BEP2D is HPV-immortalised, the lack of p53 staining is consistent with the expression of theviral protein E6. However, the extent of expression of p53 in a single tumour was modest.
Whether mutated p53 inhibits the expression of mutant p53 is unknown. In addition, Cyclin
D1 was also upregulated in tumour cells leading to the suggestion that this may circumventthe inhibitory effects of Rb signalling (Hei et al., 1996).
(E76) Non-oncogenically-immortalised human bronchial epithelial-cell systems, which canbe grown in traditional 2D culture as well as in 3D organotypic culture, are now surplanting
oncogenically-immortalised systems (Ramirez et al., 2004). These differentiated 3D cultures
were shown to have strikingly different DNA repair kinetics, particularly to high-charge, highenergy (HZE) particle exposure (Asaithamby et al., 2011). Furthermore, gene expression
analysis revealed the downregulation of multiple DNA repair pathways which would render
cells susceptible to DNA damage and the promotion of genomic instability. Such organotypic
cultures should shed new light on the molecular alterations after radiation exposure that leadto lung cancer, including the advantage of 3D growth and manipulation of genes associatedwith “stemness”.
E.4.2. Mutagenesis(E77) As described above, in view of the only recent isolation of undifferentiated lungstem cells, the consequences of radiation-induced mutations in this cell population or in theirpluripotent progenitors have yet to be analysed.

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E.5. Models of carcinogenesis bridging the stem cell concept and epidemiology

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(E78) Shown below is a list of important areas for future research:
1. Better functional analyses of putative lung stem cells (e.g. labelling, marker studies,stem-cell pool size) especially in human tissue.
2. Determine the target cells for radiation-associated carcinogenesis because this is crucial
for understanding the temporal patterns of radiogenic cancer.
3. Isolate and propagate lung tissue stem cells to investigate the differential radioresponses
between different types of stem cells.
4. Determine the location of stem cells in mouse studies: the microenvironmental influences,
positional information, dormancy, and oxygen conditions in the niche.
5. Know more about changes in stem cells related to differences and risk as a function ofage. As part of this, we need to know the increased risk for premalignant lesions as aprocess of increased age, and to determine if this increase in age-associated preneoplastic
lesions counterbalances the decline in turnover rate through ageing processes which may
decrease risk.

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ANNEX F: SKIN STEM CELLS AND RADIATION CARCINOGENESIS

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F.1. Skin carcinogenesis

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(F1) Skin cancer is separated into two categories, melanoma and non-melanoma skincancers. As there is little evidence concerning a possible link between radiation exposure andmalignant melanoma incidence, this type of cancer has not been included in this annex.
Emphasis is placed on carcinomas, because the great majority of skin cancers are of epithelial
origin.
F.1.1. Basal cell carcinoma(F2) BCC is the most frequent cancer in both men and women among populations ofEuropean ancestry, also called Caucasian populations, thus accounting for 29% of all cancers(Epstein, 2008). Its incidence appears to be increasing worldwide. In Caucasian populations,
BCC accounts for 80% and SCC for 16% of human sporadic skin cancers. Despite this highfrequency, the death rate from BCC is very low, because these tumours rarely metastasise.
BCC, which arise from epidermal keratinocytes, are mostly localised tumours. They invade
locally, where they can cause disfiguring and destruction of skin. The fact that they are slow
growing with relatively stable genomes presumably limits the accumulation of additional
DNA abnormalities that might confer a metastatic potential. Infrequently (<0.1% of cases),
metastases can develop regionally in lymph nodes or at sites distant from the primary tumour,in the lung, bone and internal organs.
(F3) The principal aetiologic factors include ultraviolet (UV) radiation and ionisingradiation, chemical carcinogens (arsenic, polycyclic aromatic hydrocarbons, psoralens) andpossible infection with papilloma viruses. Sunlight is a major environmental factor for BCC
development, as demonstrated by the fact that mutations are frequently UV specific. However,
the exact relationship is complex. The use of sunscreens has been found not to correlate with
reduced BCC risk (Green et al., 1999), even in the long term (van der Pols et al., 2006). Also,
the incidence of BCC peaks at about twofold at 10,000 to 35,000 hours total exposure, andsurprisingly, does not increase with further exposure. Several authors have postulated thatBCC development may correlate better with intermittent sunlight exposure than with total
exposure (Zanetti et al., 2006).
(F4) Although the majority of BCCs arise sporadically, genetic factors may also have asubstantial role. Several genetic diseases are associated with the development of skin cancer,
including xeroderma pigmentosum (XP) and Gorlin’s syndrome. Apart from genetic diseases,very little is known about population genetic susceptibility to BCC. In contrast to Caucasian
populations, persons of the South Asian and African ancestry with dark skin colour are
resistant to BCC development. SNP technology was used to characterise DNA sequencevariants that confer susceptibility to cancer. Some of these variants seem to operate throughtheir association with fair pigmentation traits, resulting in reduced protection from thedamaging effects of UV radiation (Han et al., 2006). Other sequence variants, such as K5,
have no obvious role in pigmentation or UV susceptibility, but instead seem to operate in thecontext of growth and differentiation of the basal layer cells of the epidermis (Stacey et al.,2009).
(F5) Concerning the molecular mechanisms of development of this cancer type, the only
known molecular abnormality for a long time was p53 mutations, which are found in 30% of
sporadic BCC. This lack of knowledge was due to the difficulty of growing human BCC in

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tissue culture or as xenografts. It was also due to the lack of an appropriate animal model,
because mice do not develop BCC, either sporadically or after exposure to UV, ionisingradiation or chemicals. Identification of a genetic event in a rare subset of patients who havea great propensity to develop BCC pointed to aberrant SHH signalling as one of the pivotal
defects leading to formation of these tumours (Fig. F.1.). This rare heritable disorder isGorlin’s syndrome [also known as basal cell nevus syndrome (BCNS)] which is caused bymutations in the patched 1 (PTCH1) gene, encoding the receptor to which the HH ligand
binds (Epstein, 2008; Hahn et al., 1996). The incidence of this syndrome ranges from
1/56,000 in UK to 1/256,000 in Italy (Lo Muzio, 2008). Aberrant activation of the SHH
pathway has been observed in sporadic and hereditary BCC. Mutations in at least one alleleof PTCH1 have been identified in 90% of sporadic BCC.

Fig. F.1. The SHH signalling pathway is frequently deregulated in BCC. Panel A. In the absence of
extracellular hedgehog (HH) ligands (SHH, Indian HH or Desert HH), PTCH1 maintains the HH
pathway in an inactive state by inhibiting the signalling effector smoothened (SMO). In the presenceof HH ligand, the positive regulators of the HH pathway are shown in green and the inhibitors areshown in grey. During physiological HH signalling, binding of SHH to PTCH1 receptor relieves thenormal repression of SMO. Then, SMO transduces the signal which leads to the activation of gliomaassociated gene homologue (GLI), transcription factors allowing transcription of various target genes.
Panel B. The HH pathway is always active in BCC in the absence of ligand, due to mutations in
PTCH1 or SMO. Evidence for a role of a deregulated control of HH signalling in the BCC
pathogenesis comes from studies of patients with Gorlin’s syndrome and mouse models in which HH
signalling is constitutively expressed. Germline mutations in the PTCH1 gene confer a strong
susceptibility to develop multiple BCC in patients with Gorlin’s syndrome, both spontaneously andafter ionising irradiation (reproduced from Lo Muzio, 2008). (Permission needed)(F6) Activation of the SHH pathway can be recapitulated by expressing the SHH protein orproteins of its signalling pathway ectopically in regenerated human skin, thereby inducing
cancer in human tissue by using only one genetic element (Fan et al., 1997). High-SHHexpressing tissue showed cardinal BCC features. These data represented the firstexperimental induction of human neoplasia by a defined genetic alteration, as well as the firstexperimentally induced malignant conversion of human tissue (Khavari, 2006). Additionally,
the surprising finding was the induction of features of the most common cancer by a single
genetic alteration that activates a dominant developmental pathway, an observation that might
help to explain how common is this cancer. Khavari’s group used a specific model, which isgrafting of in vitro reconstituted human skin in immunodeficient mice, which requires high183

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cell proliferation at several steps. This might explain why this model allows obtaining withinmonths what requires years in human skin. In vivo, in undisturbed skin with a slower turnover,
and after more moderate activation of the SHH pathway, a much longer latency period isrequired for BCC induction.
(F7) Another gene has been implicated in BCC development, the phosphatase and tensin
homolog (PTEN) gene. PTEN is a tumour suppressor for skin cancer and loss of PTEN
activity appears critical for UV-induced skin tumourigenesis (Suzuki, 2003).

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F.2. Radiation carcinogenesis in human skin

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F.1.2. Squamous cell carcinoma(F8) Similar to BCC, SCC also arises from epidermal keratinocytes, but SCC is a moreaggressive tumour that can form lethal metastases. Sunlight is clearly the major
environmental factor for SCC development. The incidence rises with the total number ofhours of sun exposure (Zanetti et al., 2006) and the use of sunscreens correlates with reduced
SCC risk (Green et al., 1999), even in the long term (van der Pols et al., 2006). Spontaneous
SCCs are associated with mutations in Ras, p53 and p16. The Ras GTPase is mutated to an
activated form in 20% of sporadic SCC. Ras is also strongly activated biochemically in its
GTP-bound form in most human SCCs through additional mechanisms besides direct Ras
mutation. Such mechanisms might include Ras gene amplification, overexpression of
upstream RTKs, or loss of inhibitory mRNAs. In addition, c-Myc overexpression andactivation of Stat signalling occur frequently in skin SCC development, notably playing a rolein the progression to the tumoural state. SCC arises most commonly as a sporadic tumour, but
it is also characteristic of several inherited disorders, including those that impair DNA repair
and genomic stability, such as XP and dyskeratosis congenita (DC), in which SCC mightarise as a secondary result of increased genomic mutagenesis. SCC also affects
approximately 75% of patients who are afflicted with recessive dystrophic epidermolysis
bullosa (EB). In summary, SCC is a skin tumour that differs from BCC in many respects,
including the aetiology, genetics, incidence rate and pathology.

F.2.1. Cancer types(F9) The first type of cancer documented as being associated with exposure to ionisingradiation was skin cancer, which was reported only 7 years after the discovery of x-rays(Frieben, 1902). Ionising radiation is a general risk factor for workers, as recently describedby the European HELIOS study performed for 10 major occupational groups (Suarez et al.,2007). Radiation-induced tumours in skin are mainly carcinomas, developing fromkeratinocytes of the basal layer of epidermis, which is the deepest layer of this multilayered
epithelium. An association between low-LET external exposures and the risk for these nonmelanoma skin cancers has been demonstrated in a series of studies, based on accidental andmedical treatment data (UNSCEAR, 2008). Some rare tumour types have also been described,
such as trichoblastoma and trichoblastic carcinomas, which are tumours with differentiation
towards hair structures. No significant relationship of melanoma with ionising radiation
exposure has been demonstrated, including in A-bomb survivors (UNSCEAR, 2000, 2008).
(F10) BCCs are the more frequent type and continue to show an approximately steady ERR
for 40 or more years after radiation exposure (Dutreix, 1986; Preston et al., 2007). Induction
has been well documented, and it has been reported after occupational, therapeutic andaccidental exposures (ICRP, 1992, 2000, 2007; Shore et al., 2002). In the survey of A-bomb

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survivors for the time period 1958-1987, a strong positive dose-response trend for BCC was
reported. The best fit dose response for BCC was non-linear, with an ERR Sv-1 of 0.7 (90%
CI: 0.1, 2.8) below 1 Sv and 4.0 (1.9-9.0) above 1 Sv (Ron et al., 1998; Charles, 2007). This
LSS cohort is very informative, because skin cancers are relatively rare in Japan, withestimated background incidence rates being 3 per 100,000 each for BCC and SCC. These
rates are much lower than in the US Caucasian population (200 for BCC and 40 for SCC).
The tinea capitis cohort studies in New York and Israel demonstrated significant BCC
development after medical irradiation of the scalp (Ron et al., 1991; Shore et al., 2002), withfurther confirmatory evidence from a large population-based case-control study (Karagas,
2007). Histopathologic review of a case series suggested that up to 23% of nominal radiationassociated BCCs might be trichoblastomas or trichoblastic carcinomas, which are tumours
with differentiation towards hair structures (Fazaa et al., 2007), although therepresentativeness of the series is unknown.
(F11) The evidence for induction of SCC by ionising radiation is much less clear than thatfor BCC. The proposed ratio of radiation-induced BCC:SCC incidence is generally 10:1, but
it varies according to the particular study. For early radiation workers, who frequently had
high and chronic exposures to the hands, there were numerous reports of SCC (Shore, 1990).
A population-based study among men in Alberta, Canada, reported a 5- to 6-fold increase inincidence of BCC and SCC associated with nondiagnostic x-ray exposure (Gallagher et al.,1996). A more recent study performed in New Hampshire found an increased risk of both
BCC and SCC in relation to therapeutic ionising radiation (Karagas et al., 2007; Lichter et al.,2000). Elevated risks were confined to the site of radiation exposure (BCC odds ratio, 3.30;
SCC odds ratio, 2.94). One possibility is that SCC can be induced significantly only by highradiation doses (Charles, 2007). A mortality rate of 1% was evaluated for SCC (ICRP, 1992).
(F12) Radiation-induced skin tumours are very similar to sporadic skin tumours. Ionisingradiation appears essentially to increase the natural rate. Although the nominal risk
coefficient for skin cancer appears to be the highest among all tissues (Table 1.1.), the healthdetriment is much smaller for skin cancer than other radiation-induced cancers, and hence the
ICRP has assigned a small value of tissue weighting factor (wT) for skin of 0.01. Further
details of this are as follows. As described in the ICRP Publication 103, the nominal risk
estimates (which are excess rates, and not ERRs) for most of the tissue sites considered, were
determined by the application of LSS-based radiation risk (ERR and EAR) estimates to rates
in hypothetical “composite” Asian and Euro-American populations. However, the nominal
coefficient for skin cancer was not determined in this way. Instead (as noted in Publication
103, paragraph A118), skin cancer was handled differently. In particular, the nominal skincancer incidence risk coefficient was chosen to be consistent with the skin cancer mortality
risk coefficient in Publication 60, which uses a nominal skin cancer mortality risk coefficientof 2 deaths per 10,000 people at 1 Sv and also indicates (Table B-19 in Publication 60) thatthe lethality fraction is 0.002. This translates into the nominal skin cancer incidencecoefficient of 1,000 cases/104/Sv given in Publication 103 for a whole population (the value
used for a working age population is 670, Table 1.1). This nominal risk coefficient was notderived explicitly based on LSS results. Despite the large nominal incidence risk for skincancer, the tissue-weighting factor for skin cancer is the same (0.01) in Publications 103 and60. The reason for this is that radiation-associated skin cancer cases are given a very low
weight in the computation of detriment. Regarding the present report on stem cells as target
cells for induced cancers, the implication of the above discussion is that skin stem cells havea high sensitivity to carcinogenesis, but this has a large uncertainty as deduced from currently
available publications.

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F.2.1. Threshold dose(F13) The traditional view was that a threshold dose exists for radiation-induced skin cancer,
in the range of 8 to 10 Gy. In a cohort of radiotherapy patients (Lichter et al., 2000), the RR
of developing skin carcinoma after receiving therapeutic ionising radiation was increased for
total radiation doses higher than 30 Gy, which was consistent with other previous estimates
for the radiation dose necessary to induce skin carcinoma after therapy protocols (using
multiple doses of 2 Gy per fraction). The risks of BCC and SCC were increased specificallyamong those treated with 10 Gy per week or less, and less than or equal to 2 Gy per treatment.
Surprisingly, more-highly-fractionated doses of ionising radiation, involving lower doses per
fraction, appeared to be more carcinogenic than the classical protocols. In a study of skincancers induced by radiotherapy in childhood, BCCs were strongly induced within theradiation field by (total) therapeutic doses ranging from 7 to 27 Gy (Levi et al., 2006a). No
case of cutaneous SCC, or of malignant melanoma, was observed. In 6 different studies of theeffects of medical exposures, where mean doses ranged from 2.25 to 6.8 Gy, the mean RR
per Gy was 1.6, ranging from 1.1 to 2.1 (Shore, 2001). Other available data suggest that
relatively small doses do have an effect. In the A-bomb cohort where the mean dose was <0.3
Gy, analyses of total non-melanoma skin cancer which allowed for a spline (i.e. a change inslope) at 1 Gy fitted the data better (P = 0.005) than a pure linear model (Preston et al., 2007).
Using the spline model, the estimated ERR per Gy was 0.17 (non-significant) for doses below
1 Gy and 1.2 (90% CI 0.57; 2.3) for doses above 1 Gy. An analysis of skin BCC found an
ERR Gy-1 of 2.0 (95% CI 0.7, 4.3) above about 0.6 Gy, but no dose trend below that (slope =-0.05) (Sugiyama, 2012). Thus, the A-bomb survivor data indicate that skin cancer, BCC in
particular, can be induced by acute exposure at moderate doses above about 0.5-1 Gy, withlittle skin cancer risk at lower doses, so that there could be a threshold dose of ≤0.5 Gy.
F.2.2. Variation risk by age at exposure(F14) Several studies have examined how risk varies by age at radiation exposure. In the Abomb study, there was a 30-fold variation in ERR for BCC with age of exposure (Ron et al.,1998). The ERR was 21 for those aged 0-9 years at exposure and 0.7 when exposed at ages
higher than 40. In a later analysis, the ERR also decreased rapidly with increasing age at
exposure (P <0.001), but there was little change with attained age (P >0.5) (Preston et al.,2007). In the case of tinea capitis, both an Israeli study (Ron et al., 1991) and a New York
study (Shore et al., 2002) showed clear age effects. The declines in risk amounted to 11% peryear of age at exposure in the A-bomb study, 13% in the Israeli study and 13% in the New
York study. A similar strong relation between BCC induction and radiation exposure inchildhood was also shown in a study of secondary skin neoplasms in long-term survivors
from cancer (Levi et al., 2006b; Schwartz et al., 2009; Watt et al., 2012). Thus, the available
studies show that skin cancer risk is greater from radiation exposure at younger ages than atolder ages (Shore, 2001).
F.2.3. Interaction with UV(F15) An interesting biological question is whether UV and ionising radiation have aninteractive effect on skin tumour induction. The initial study of A-bomb survivors concluded
that the ERR per Sv was higher on UV-shielded skin than on the face and the hands (Ron etal., 1998). A more recent study of the same cohort showed that the EAR of BCC per unit skinsurface area, related to radiation exposure, did not differ between UV-exposed and shielded186

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parts of the body, suggesting a simple additivity of the radiation-related and background BCC
risks (Kishikawa et al., 2005). Mizuno et al. (2006) assessed the association with UV in BCC
from the Japanese cohort, by characterising mutations in the p53 gene. They showed that
70% of BCC had p53 mutations independent of ionising radiation or UV. However, thedistribution of mutation types depends on the UV or ionising radiation exposure, suggesting
different modes of action of UV and ionising radiations in inactivating the gene.
(F16) After medical exposure, studies of patients irradiated for tinea capitis reported
contradictory results, with RR greater on the relatively UV-shielded scalp than on the faceand neck (Ron et al., 1991), or greater for the UV-exposed margin of the scalp (21/100 cm2
per Gy) than for the (relatively) UV-shielded scalp (4.7/100 cm2 per Gy) (Shore et al., 2002).
In a study of second skin neoplasms in patients surviving from childhood cancer, all the
BCCs were located within the radiation field, thus indicating that ionising radiation is the key
aetiological factor, even in the absence of any meaningful interaction with UV (Levi et al.,2006b).
(F17) Thus, no clear evidence appears after accidental or medical exposure for aninteraction between ionising radiation and UV exposure. However, there is some evidence foran association of ionising radiation sensitivity and UV sensitivity. The New York study ofionising irradiation for scalp ringworm found a substantial excess of BCCs among whitepatients but no skin cancer among the irradiated black patients (Shore et al., 1990). The
radiation risk for skin cancer was 10 times higher among the Caucasians. For SCC, anassociation with radiotherapy was principally observed among persons with a sun-sensitive
phenotype (Lichter et al., 2000). Finding few excess skin cancers among irradiated AfricanAmericans as compared to Caucasians with a comparable dose, may indicate that skinsusceptibility to UV exposure increases the excess risk from ionising radiation.
(F18) UNSCEAR (2000) considered that the question of a possible interaction between
ionising and UV radiations remained unresolved, and that more data were needed to fully
understand this complicated relationship.
F.2.4. Gender role(F19) Male gender is a known risk factor for sporadic BCC in humans and is associated withmore BCC than in females (rate ratio: 1.2). Although the role of estrogens in the developmentof skin cancer is controversial, the results point to an effect of hormonal status on BCC
development. However, for radiation-induced skin cancer, none of the major studies has
found a difference in cancer risk according to gender (Shore, 2001). In the A-bomb survivor
cohort, no gender role was found in the initial description of skin cancer (Sadamori et al.,1989). This was confirmed in more recent studies on BCC. The incidence of BCC in sunexposed sites was significantly higher in men than in women, but no gender effect wasobserved in the incidence of radiation-induced BCC at non-exposed sites among the survivors(Kishikawa et al., 2005; Naruke et al., 2009).
F.2.5. Genetic susceptibility to radiation-induced skin cancers(F20) The relationship between ionising radiation exposure and cancer risk varies greatly
among the different human cancer-prone genetic diseases. For ataxia telangiectasia (AT)patients, where cells exhibit a higher sensitivity to ionising radiation, no excess of radiationinduced skin cancer has been described. For XP, a genetic disease exhibiting hypersensitivity
to UV radiation and a high incidence of skin cancers, no excess for radiation-induced skincancer has been described. The DC syndrome is a very rare telomere biology disorder, with a187

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high risk of SCC induction. Both hypersensitivity (Cengiz et al., 2004; M'Kacher et al., 2003)
and cancer induction by ionising radiation (Hyodo et al., 1999) have been described for thissyndrome.
(F21) Evidence for a correlation between radiation exposure and increased risk in skincancer due to genetic susceptibility comes mainly from studies of patients affected by
Gorlin’s syndrome (or NBCC) (Lo Muzio, 2008). This rare autosomal dominant disease ischaracterised by a wide range of developmental abnormalities and a predisposition todeveloping tumours, particularly BCC. In Gorlin’s patients, BCC usually starts at puberty,
and up to 90% of patients will have developed a BCC at the age of 40 years. For black
Gorlin’s patients, the incidence of BCC is lower (40%) and occurs at later ages, probably
linked to protection due to skin pigmentation. After radiotherapy for medullablastoma inchildren with Gorlin’s syndrome, multiple BCCs were found in the radiation field at 3 to 6
years after irradiation (Kleinerman, 2009; Mitchell et al., 2005; O'Malley et al., 1997).
Fibroblasts isolated from the skin of three Gorlin’s patients were described as sensitive toionising radiation (Chan and Little, 1983). Data from Gorlin’s syndrome probably point todifferences between UV and ionising radiation, as Gorlin’s skin keratinocytes are not
hypersensitive to UV-induced cell death (Brellier et al., 2008).

7746	
  

F.3. Radiation carcinogenesis in rodent skin

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(F22) Limitations of mouse models. Human and mouse differ in their skin organisation andhomeostasis (Khavari, 2006). One example is hair follicle biology. Architecturally, theepithelium in adult, fur-covered murine skin is disproportionately comprised of denselydistributed hair follicles. In contrast, epithelium in human skin is proportionately much moreinter-follicular (mean of 20 follicles per cm2). Moreover, murine hair follicles undergosynchronous cycles that span the first 2 months of life, a time period when many neoplasia
phenotypes are either initiated or studied, and the oscillation of follicular cycles can havedramatic effects on the proliferation of the cutaneous epithelial pool (Stenn and Paus, 2001).
This situation differs from human skin, in which sparse hair follicles generally cycle
asynchronously throughout life. Another example is epidermis thickness, around 25 µm with
2-3 cell layers in mice and 100 µm with 6-10 cell layers in humans, which results in greater
percutaneous absorption and decreased barrier function in mice.
(F23) As a consequence of these differences, mice differ from humans in a number ofgeneral ways that are relevant to cancer. First, the spectrum of malignancies differs
dramatically (Anisimov et al., 2005; Hahn and Weinberg, 2002; Khavari, 2006; Rangarajan etal., 2004; Rangarajan and Weinberg, 2003). Whereas most human tumours arise in epithelia,
most murine tumours occur in the form of non-epithelial sarcomas and lymphomas.
Epidermal skin cancers are very rare in these animals when they have not been exposed to
UV or ionising radiation, which is different from the high basal rates seen in humans, notably
from European ancestry. Moreover, all classical mouse skin carcinogenesis models readily
produce tumours of the squamous type, but none of the basal type. Second, the origin of
epidermal tumours is the interfolllicular epithelium for most human skin cancers, whereas
most carcinomas are from hair follicle origin in mice. Third, there are species-specificdifferences in the mechanisms of cell transformation. Telomeres are approximately five times
as long as human telomeres, a difference that might explain the ease of immortalising mouse
cells compared with human cells (Rangarajan et al., 2004). Differences in oncogene
signalling are illustrated by the action of oncogenic Ras through the Raf downstream effector
cascade in murine fibroblasts, whereas the oncogenic function of Ras takes place through Ralpathway induction in human cells (Hamad et al., 2002).

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(F24) These substantial cross-species differences argue for caution in applying data fromstudies of murine neoplasia directly to humans. Murine models suffer from various
limitations, paramount among these being the uncertain applicability to cancers that arise in
humans. This is also true when ionising radiation is the carcinogen. For example, BCC is the
major skin tumour induced in Caucasian human populations, whereas in mice, ionisingradiation readily produces SCC but no BCC. Rats may represent a better model than mice, atleast concerning the type of tumours. In rats, epithelial tumours develop more frequently than
mesodermal tumours (Anisimov et al., 2005). After exposure to either single or multiple
doses of ionising radiation, the most frequent type of skin tumour in rats is BCC, followed by
SCC (ICRP, 1992). However, the radiation had to penetrate at least about 180 µm to induce
tumours, demonstrating that the main target was also the hair follicle (Albert et al., 1967).
(F25) Cancer genes. The mechanisms of development of skin tumours after radiation
exposure are still largely unknown. It is clear that ionising radiation can act as an initiator, apromoter and a complete carcinogen. In rats, c-myc appears to play an important role, as
activation of c-K-ras and c-myc oncogenes was found in SCC (Sawey et al., 1987), as well as
amplification of c-myc during BCC and SCC progression (Garte et al., 1990).
(F26) Cancer gene manipulation is a field where mouse studies may be important, due to thepower of gene-knockout technology in that species. Recently, the discovery of the role of thepatched gene in the patients with Gorlin’s syndrome stimulated the engineering of several
mouse models, in which HH signalling could be manipulated and BCCs could be produced.
These models have allowed more basic investigations into BCC tumourigenesis, including
after ionising radiation. Ptch1 codes for a transmembrane protein that works as anantioncogene by negatively regulating the SHH pathway and cell proliferation. The group of
Anna Saran observed BCC induction in irradiated Ptch1+/- mice. Unirradiated mice develop
precursor lesions, that progress to BCCs only in irradiated mice. Ptch1neo67/+ mice and wildtype littermates of both sexes were whole-body irradiated with 3 Gy of x-rays as adults (age 3
months). In these mice, BCC incidence was 12%, whereas no BCCs were observed inunirradiated mice. In addition, 2-month-old mice were subjected to local irradiation of thedorsal skin with a single x-ray dose of 4 Gy. These mice showed a 4.9% BCC incidence(Mancuso et al., 2004; Pazzaglia et al., 2004).
(F27) To test for interactions between SHH signalling and poly (ADP-ribose) polymerase 1(PARP-1), a major protein of the repair of DNA strand breaks, PARP-1-null mice werecrossed to Ptch1 heterozygous mice. Double-mutant mice were strikingly more susceptible to
BCC, with over 50% of animals developing multiple, large, infiltrative tumours within 30
weeks of age. The results provide genetic evidence that, in mice, DNA-strand-break repair
controlled by PARP-1 is required and cooperates with the SHH pathway to prevent BCC
formation in response to DNA damage (Tanori et al., 2008).
(F28) Also, using cell-fate tracking of x-ray induced BCCs in Ptch1+/- mice, their essentiallyexclusive origin was found to be K15-expressing stem cells of the follicular bulge (Wang etal., 2011). However, conditional loss of p53 not only enhanced BCC carcinogenesis from thebulge, but also produced BCCs from the interfollicular epidermis, at least in part, byenhancing SMO expression. This latter finding is consistent with the lack of visible tumours
on ears and tail, sites lacking SMO expression, in Ptch1+/- mice.
(F29) Gender role. The gender role has been assessed in the Ptch1neo67/+ mouse model(Mancuso et al., 2004). Although microscopic basal cell lesions were observed in males andfemales, infiltrative BCCs only developed in males, showing a dramatic gender role in thatmodel. The role of endogenous estrogen in that difference was addressed in Ptch1+/- mice,
useful for BCC studies, and skin carcinogen-susceptible (Car-S) mice, elective for studies ofpapilloma and SCC induction. Susceptibility to radiation-induced BCC or chemically189

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induced SCC was dramatically increased in ovariectomised Ptch1+/- and Car-S females andrestored to levels observed in males. These findings underscore a highly protective role ofendogenous estrogen against skin tumourigenesis in two independent mouse models of skincancer (Mancuso et al., 2009).
(F30) Type of exposure and radiation quality. Experimental results suggest that reduction indose rate does reduce the carcinogenic effect to some degree. The data of Hulse et al. (1983)and Papworth and Hulse (1983) suggested an acute threshold of about 16 Gy for induction of
epidermal tumours in rats. The resistance to the induction of SCCs by protracted irradiation
was well illustrated by the finding that 0.75 Gy given three times a week over their lifetime
induced only a 3% incidence of SCC, whereas higher doses per fraction resulted in a 100%incidence. The LQ dose-response relationship for the induction of skin tumours in ratssuggests that fractionating the exposures or lowering dose rate should reduce theeffectiveness.
(F31) Burns et al. (1975) reported that the tumourigenic effect was decreased by splitting adose and the reduction in effect increased with increasing time between the exposures. Split
doses of 0.7 MeV electrons were used by Burns and Vanderlaan (1977) to study the repair
and recovery of the carcinogenic events and found a repair half time of about 3 hours. The
ability to repair the initial events was maintained for many exposures. The data for multiple
daily exposures when plotted as tumours per rat at 48 weeks as a function of dose on a loglog scale gave a slope of 2.4 which is in approximate agreement with the D2 term in the LQ
model. The increased exponent for multiple doses delivered over a lifetime was much greaterthan expected if the effects of each individual dose were simply additive in each timeincrement. In the multistage theory of carcinogenesis, the increased exponent is interpretable
either as an increased number of events occurring stochastically in time or as clonal growthof one or more of the intermediate stages (i.e. of mutated stem or progenitor cells in thelineage) (Burns and Albert, 1986b). There was also a direct effect of age, with older rats
being less susceptible to tumour induction, but in contrast, the half time for persistence of
carcinogenic damage became shorter, being 3.6 hours for 28-day-old rats, 1.2 hours for 112day-old rats and 0.08 hours for 182-day-old rats (FJ Burns, personal communication). The
use of different electron energies and penetrations identified the target cells as being at 0.30.4 mm depth, irrespective of the follicle size changes throughout the hair growth cycle(Burns et al., 1976).
(F32) The addition of solar spectrum UV radiation, of which 80% was absorbed in theepidermis, yielded more tumours after lower doses of ionising radiation but fewer tumours
after higher doses. The reduction seemed to be a sterilising effect on the development ofsmall tumours, because when the UV exposures were stopped, tumours began to appear atabout the same rate as observed earlier in the groups that received ionising radiation without
subsequent UV. Interestingly, the UV prevented the onset of all types of tumours including
SCC and hair follicle tumours. If the sterilising effect was a result of direct action of the UV,
it would be necessary to conclude that the presumptive tumour cells were in or just below thesurface epidermis at the time of UV exposure (Burns and Albert, 1986a). This would
implicate both this region and the region at 0.3 mm together in the carcinogenic process.
There are various questions that remain to be answered: e.g. (1) Do the stem cells become
more quiescent with increasing age and hence may have more time for efficient repair?; (2)Was the time-power/slope increase in tumour incidence with multiple exposures over a
lifetime a result of radiation dose to cells that had already embarked on neoplastic pathways
as a result of prior exposures?; (3) Are the target cells for carcinogenesis restricted to earlystem cells or including daughter progenitor cells?; and (4) How important in tumour
progression are radiation effects on the stem cell niche?
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(F33) In the mouse, it is necessary to protract or fractionate the dose to obtain a dose that is
sufficiently high to produce SCCs without excessive damage to other tissues. However, in theexperiments of Coggle and Williams (1990), no reduction in effectiveness was found over a
1000-fold range of β-radiation doses down to 0.1 Gy/min. In contrast, Hulse et al. (1983) did
find a reduction from a total of 30 tumours to 5 in comparable numbers of mice when thedose rate was reduced from 1.1-2.0 Gy/min to 0.017-0.024 Gy/min. Neither the dose rate nor
the total dose were low in terms of radiation protection or carcinogenesis in the experimentsof Hulse et al. (1983) and Williams et al. (1986). Thus, it is possible, as the experiments ofHulse et al. suggested, with very low dose rates, say 0.01 Gy per day, the reduction in
effectiveness might be considerable.
(F34) There is a split-dose sparing effect on the survival of irradiated macrocolony-forming
cells in mouse epidermis, irradiated using high doses (see Fig. F.6.), and hence dose-rate
effects on clonogenic survival are expected.
(F35) Regarding LET, studies reviewed by Burns and Albert (1986a, b) indicated that in rats
exposed to single doses, epithelial tumours appeared in about 10 weeks post irradiation, andthen at an increasing rate over a period of at least 80 weeks (Burns and Albert, 1986a, b).
With exposures to monoenergetic electrons (0.34 keV/µm) that penetrate at least 1 mm thedata for the dose response for skin tumours/rat at 80 weeks as a function of dose could be
fitted using a quadratic model, whereas irradiation with argon ions (125 keV/µm) resulted in
an approximately linear dose response up to about 9.0 Gy. The authors concluded that theresults were consistent with an LQ dose-response model with the linear component dependent
on the LET of the radiation. The types of skin cancer induced in rat skin were independent of
LET, and RBE values of around 5 were reported at the lowest dose of 1 Gy low-LET
radiation where tumours were induced. The question of the RBE for skin cancer induction by
high-LET radiations was stated by ICRP (1992) to be essentially unanswered, and the topic
was further reviewed by Masse (Masse, 1995).

7900	
  

F.4. General features of skin

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(F36) The skin is the largest organ of the human body. Adult skin comprises between 15 and
20 % of the total body weight, with a surface area of 2 m2. For the average adult human, theskin is between 2-3 mm thick. Skin performs numerous functions, including protection,
sensation, heat regulation, excretion, and absorption. It consists of two broad tissue types: theepidermis – an external stratified, non-vascularised, epithelium; and an underlying connective
tissue called the dermis – consisting of dense fibrous components produced by fibroblasts.
The dermis is usually less than 2 mm thick and houses many of the skin’s functional
components, including its vascular, neural and lymphatic systems and its multiple epidermal
appendages. The latter include excretory and secretary glands (sebaceous, eccrine andapocrine glands), keratinising structures (hair follicles and nails), and sensory nerve receptors.
Finally, anatomists include a third skin layer, the subcutis or hypodermis, consisting of fatty
connective tissue that connects the dermis to underlying skeletal components. As the majorityof skin cancers have an epithelial origin, this section focusses on epidermis and keratinocytes.
F.4.1. The epidermis(F37) The epidermis is the outermost layer of the skin. It forms the waterproof, protective
wrap over the body surface and is made up of a stratified squamous epithelium with anunderlying basal lamina. The total thickness is usually between 20 to 80 µm thick, depending
on body localisation, going up to 1.4 mm on palms and soles (Potten, 1986). The epidermis of191

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a single human being comprises approximately 8 x 1010 basal keratinocytes (Larcher et al.,2007).
(F38) The epidermis consists of four main types of cells: keratinocytes, melanocytes,
Langerhans cells and Merkel cells. Keratinocytes, the cells that make the keratin proteins, arethe predominant type of cells in the epidermis (Fig. F.2.). At the lowermost portion of theepidermis, named the basal layer (stratum germinativum), are immature, rapidly dividing
keratinocytes. As they mature, keratinocytes flatten out and move upward to the suprabasal
layers. At the end of their life cycle, they reach the uppermost layer called the horny layer(stratum corneum). This layer consists mainly of dead keratinocytes, filled with keratins andlipids forming a protective barrier. Dead cells from the stratum corneum continuously slough
off through the desquamation process and are replaced by new cells coming from below. The
human epidermis completely renews itself every 3 to 5 weeks. Two to 3 billion skin cells areshed daily by the desquamation process. The body expends this effort to replace epidermis
every month, because the skin constitutes the first line of defense against dehydration,
infection, injuries and temperature extremes.
(F39) The salient features of the structure of the epidermis can be summarised as follows(ICRP, 1992):(1) The epidermis is composed of viable and non-viable layers. The outer layers of deadcells, the stratum corneum, constitute ~25% of the total epidermal thickness;(2) In the viable epidermis, stem cells are restricted to the basal layer, predominantly
towards the bases of rete pegs, although cell divisions do occur in suprabasal cells;(3) More than 50% of basal cells are to be found at a depth of >200 µm, distributed in theshaft of hair follicles at varying depths within the dermis; and(4) The depth of the basal layer in the interfollicular epidermis varies greatly but is between
20-100 µm in most body sites. On the hands, the epidermis of the fingertips is thicker
and the depth of the basal layer is >160 µm.
(F40) As described in ICRP (1991), the stratum spinosum is made up of a variable numberof cell layers. The cells of these layers are rich in cell-to-cell contacts, desmosomes, whichbind the cells firmly together. It is the structural and proliferative organisation of cells in thebasal layer and the stratum spinosum which largely determines the response of the epidermis
to radiation-induced injury. The basal layer, which is separated from the dermis by abasement membrane, is considerably undulated, and in many regions, distinct rete ridges orpegs can be seen.
(F41) The results of radiolabelling experiments with 3H-thymidine demonstrated thepresence of labelled suprabasal cells in human epidermis (Epstein and Maibach, 1965;
Weinstein and Van Scott, 1965). In a quantitative study (Penneys et al., 1970), it was foundthat 32% of labelled nuclei were in a suprabasal position. The precise kinetic relationships
between the proliferating cells of the basal and suprabasal layers have not been established;however, a number of models have been proposed which have been linked with the presenceof the rete pegs or ridges.
(F42) Hume and Potten (1979) considered three possible models for the cell kinetic
organisation of a thick epidermis as is seen in man. In all three models, it was assumed thatthe rate of cell desquamation from the surface of the stratum corneum overlying the rete pegs
was equal to that from the viable epidermis between the rete pegs. In the model favoured by
Hume and Potten (1979), because it was in better accord with the available cell kinetic data, it
was proposed that only a small proportion of the keratoblasts in the basal layers are stem cells,with the majority of basal cells being proliferative transit cells. The stem cells are confined tothe base of the rete pegs: therefore, the transit cells increase in age with distance from thebase of the rete peg. Consequently, the proliferation indices would be highest in the region of192

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the rete pegs. In man, the exact relationship between the 68% of labelled cells located in thebasal layer and the majority of the remaining labelled cells found in the first suprabasal layer
remains uncertain. The latter may represent transit cells that still retain division capabilities.
(F43) Because of the complexity of the thick epidermis found in man, the stacking of cells
into columns (as found in rodents) may not be precise or may be totally absent in someregions (Bergstresser and Chapman, 1980; Blair, 1968; MacKenzie, 1975). Where cellstacking has been demonstrated in human epidermis, it was limited to the stratum corneum
and could not be traced beneath the granular layer (MacKenzie et al., 1981). In this respect,
the organisation of a thick human epidermis is very different from the highly organised
structure in the epidermis of rodents. In rodents, it has been proposed that each clearly
defined column of cells has its own slow cycling stem cell and a population of cells which
provide amplification of cell division. Such a cluster of cells was termed an epidermal
proliferative unit (EPU) (Potten and Allen, 1975).
(F44) If an EPU exists in human epidermis, possibly based on the rete peg, then it would
differ in many respects from the EPU postulated for a thin epidermis, such as that of thedorsum of the mouse. The rete peg unit is much larger than an EPU. In addition, the verticalorientation of a high proportion of mitoses precludes the precise sequence of an age-related
cell migration as predicted by the EPU model. Another prominent difference between the two
proposed units is the presence of suprabasal proliferative cells in the rete peg unit. If a fixed
number of cell divisions take place in the transit-cell compartment, as is the case in the EPU
model, the rete pegs would be regularly spaced; this does not appear to be the case inhistological sections of the epidermis.
(F45) The epidermis contains no blood vessels, and is nourished by diffusion from thedermis. The epidermal homeostasis and the differentiation programme are regulated bydiffusible factors regulating proliferation and differentiation, provided by the mesenchyme,
by other neighbouring tissues or by the circulation. Skin epithelium also includes appendices,hairs, sebaceous and sweat glands, which are mainly located in the dermis.

horny layer

granular layer
Epidermis
spinous layer

basal layer
Dermis

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Fig. F.2. Organisation of human epidermis (courtesy of MT Martin, CEA, France). (permission
needed)193

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(F46) The epidermis self-renews every 28 days through keratinopoiesis. It is organised infour main layers representative of the successive steps of keratinocyte differentiation.
Proliferation of keratinocytes is mainly restricted to the basal layer. By moving to the upper
layers, cells stop dividing and progressively develop terminal differentiation. Corneocytes are
eliminated in the horny layer by desquamation.
(F47) The hair follicle. Hair is a keratinised protein filament that grows through theepidermis from follicles deep within the dermis. It has two distinct parts, the hair follicle andthe hair shaft. The bulbar region of the hair follicle contains a pool of relatively
undifferentiated epithelial cells, termed matrix cells. During the “growing phase” (anagen) of
the hair cycle, these matrix cells proliferate extremely rapidly with a doubling time of 18-24
hours. This proliferation appears to be tightly controlled by the dermal papilla. After a periodof active growth in anagen, matrix cells cease to divide, and the lower follicle regresses
during catagen, which is a regressing phase. When regression is completed, the follicle enters
telogen, a resting phase that can last for several months. The matrix cells then resume
proliferation and produce a new hair bulb, thus reentering anagen and completing a hair cycle(Paus and Cotsarelis, 1999). The distribution of basal cells between the epidermis and thefollicular epithelium has not been determined for human skin.
(F48) Turnover rate. Many of the reports of cell kinetic studies in human skin are, to agreater or lesser extent, less reliable than those obtained from studies in laboratory animals,
because of the difficulties associated with the application of the experimental techniques asapplied to man. These findings were extensively reviewed by Potten et al. (1987), where it
was indicated that the majority of the reports dealt only with the basal layer of the epidermis.
Mitotic index (MI) values vary greatly, both between individual subjects in a study andbetween studies. The few estimates of the time for mitosis suggest 1.0-1.5 hours. Studies
involving in vivo labelling with 3H-thymidine have produced values for the LI of 3.8-8.1%
although many authors suggest values of between 5% and 6%. Estimates of the DNA
synthesis time (TS), using in vivo double labelling, indicate values of between 7.0 hours and10.6 hours. In a major study in human skin using the fraction of labelled mitosis (FLM)
technique, Weinstein and Frost (1969) estimated TS to be 16 hours from an FLM curve thatwas fitted by eye. This was revised to 12.1 ± 6 hours following a retrospective computermodel fitted to the same data (Potten et al., 1985). From the same analysis, the duration of
TG2 + TM was estimated to be 8.7 ± 4.6 hours. Estimates of the cell production rate (k) for
human epidermis, based on MI and LI data have varied from 5.1-8.8 cells/1000 basal
cells/hour (Potten, 1975). Values for the turnover time of the basal cell population (TT) can
also be calculated using estimates of TS and LI.
(F49) Estimates of the cell cycle time (TC) for the basal cells of the normal epidermis havecome from a number of sources. On the basis of the time elapsed between the first and second
peak in an FLM curve, obtained from in vitro studies, a value of 59 hours was proposed(Chopra and Flaxman, 1974). Values of between 50 hours and 137 hours were reported using
flow cytometry (Bauer et al., 1980; Bauer and Grood, 1975). The cell cycle of human EpiSCs
is expected (by analogy with mouse data) to be longer than that for other basal cells, but noaccurate values are known.
(F50) Age and gender specificity of tissue turnover. The above features of the tissue
architecture and the turnover rate are dependent on age and gender, and this will be addressedin this paragraph. The age periods to be discussed include those of fetal development,childhood growth and adulthood. Thus, the tissue turnover rate and the relevant informationin fetus, infant, young adult and adult are discussed. Of particular interest is the decline of theturnover rate through ageing process which contributes for the age-dependent decrease of the
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sensitivity to radiation carcinogenesis. Aged epidermis is less proliferative than young
epidermis, as exemplified by slower wound healing. However, it is not known whether
quantitative and/or qualitative alterations in the stem and/or TA compartments are
responsible for the decreased proliferation. Earlier studies found a normal or decreased
frequency of putative EpiSCs with ageing. Using long-term repopulation in vivo and colony
formation in vitro, it was shown that although no significant difference was detected in
EpiSC frequency with ageing, TA cell frequency was increased (Charruyer et al., 2009).
Moreover, aged TA cells persisted longer, whereas their younger counterparts had alreadydifferentiated. Underlying the alteration in TA cell kinetics in the aged was an increase in the
proportion of cycling keratinocytes, as well as an increase in cell cycle duration. In summary,although no significant difference in EpiSC frequency was found, TA cell frequency wasincreased (as measured by in vivo repopulation, growth fraction, and colony formation).
Furthermore, the proliferative capacity (cellular output) of individual aged EpiSCs and TA
cells was decreased compared to that of young cells. Although longer cell cycle duration
contributes to the decreased proliferative output from aged progenitors, the greater number ofTA cells may be a compensatory mechanism tending to offset this deficit. Flow cytometricmeasurements of the DNA content were performed on a large number of skin biopsies by anautomated technique. Expressed as a percentage of all viable cells in the epidermis, thefigures for cells in S-phase averaged 1.8% and for G2M 0.9%. No significant differences due
to sex were found. Concomitantly with age, the ratio S/G2M (representing the duration of S tothe duration of G2M) increased. Also, seasonal effects were clear, showing higher values for
S and G2M in June compared to November and December. Lastly, small differences were
found dependent on body-site, the ratio S/G2M being greater in legs than in arms.
F.4.2. Skin stem cells(F51) Two types of skin stem cells: Specific properties have been proposed for the
keratinocyte stem cells (KSCs) as compared to more differentiated keratinocytes. KSCs canbe defined as:
 rare cells in the skin and the only permanent, possibly anchored, long-term residents;
 found in well-protected niches; cells on which the entire lineage and ultimately the tissue are dependent, through theirself-renewal potential; they are responsible for the long-term maintenance of skin; undifferentiated cells both structurally and biochemically (cytoplasm filled withribosomes and devoid of keratin filaments);
 slow cycling in homeostatic conditions; LRCs as a consequence of their slow cycling and their specialised DNA segregation
process; and capable of very high proliferation in response to wounding and to certain growth stimuli.
(F52) Follicular stem cells. Hair follicles are self-renewing structures that cycle andreconstitute themselves throughout life, through the activity of follicular stem cells. In rodent
hair follicles, it was demonstrated that LRCs, assumed to be slow cycling KSCs, localise to aregion of the outer root sheath surrounding the rodent hair shaft termed the bulge (Cotsarelis
et al., 1990). The bulge approximates the attachment site for the arrector pili muscle, andmarks the bottom of the permanent portion of the follicle during cycling. Others argued forthe localisation of stem cells in the upper half of the follicle: x-irradiation destroys the hairmatrix, but cells in the outer root sheath can regenerate a complete hair bulb, lending strong
support to the notion that the germinative source of each generation of hair follicles must
reside in the outer root sheath and not in the bulb. Besides, the lower half of rat (vibrissa) hair
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follicles can be surgically removed, and a new hair bulb can regenerate in response to theimplantation of a new dermal papilla (Cotsarelis et al., 1990).
(F53) Using a specific model, the whisker follicle of the rat, the group of Barrandon
demonstrated at the single-cell level that cultured stem cells retain their long-term potential toform hair follicles when grafted into athymic mice (Claudinot et al., 2005). They showed that(i) clonogenicity is an intrinsic property of the adult stem cells of the hair follicle; (ii) after
cultivation for >140 poplulation doublings (PDs), these stem cells, transplanted to the dermoepidermal junction of newborn mouse skin, form part or all of the developing follicles; (iii)the stem cells incorporated into follicles are multipotent, because they generate all of thelineages of the hair follicle and sebaceous gland; and (iv) thousands of hair follicles can begenerated from the progeny of a single cultulred stem cell. Thus, accumulated evidence
confirmed that in rodents, the bulge is the repository of multipotent stem cells that support
hair follicle cycling and can repopulate interfollicular epidermis and sebaceous epithelium.
(F54) More recently, new data revealed a diversity amongst follicular stem cells that waspreviously unrecognised (Watt and Jensen, 2009). The rodent follicle now appears to be acontinuum of at least five different populations, each with specific markers, including:
1. Cells of the junctional zone, which are positive with leucine-rich repeats andimmunoglobulin domains 1 (Lrig1) and the cell-surface marker MTS24 (Watt and Jensen,2009);
2. MTS24+/CD34-, integrin α6low cells of the upper isthmus (Watt and Jensen, 2009);
3. CD34+/K15+ cells of the bulge, also LRCs (Blanpain et al., 2004; Lyle et al., 1998;
Trempus et al., 2003);
4. Lgr5+ cells, under the bulge (Jaks et al., 2008); and5. Lgr6+, above the bulge (Snippert et al., 2010).
Surprisingly, some of these populations (populations 2 and 4 in the above list) are actively
cycling. The specific roles of all these populations remain to be determined. However, thebulge population still remains the major stem cell repository.
(F55) Regarding the stem cell niche, Wnt/β-catenin is required for follicle SC maintenanceand niche biology, and β-catenin activation is essential for promoting quiescent follicle SCs
to proliferate and terminally differentiate along the hair cell lineage (Yang and Peng, 2010).
Further, β-catenin stabilisation promotes de novo HF morphogenesis, and constitutively
active β-catenin expression results in pilomatricoma. Both BMP and TGFβ signals are
required for quiescent niche maintenance: BMP deletion results in SC activation, whereas
TGFβ may play a role in SC identity maintenance.
(F56) Concerning human skin, although several lines of evidence have suggested that thehair follicle also provides a niche for KSCs, anatomic boundaries, biochemical
distinctiveness, and global gene expression pattern are ill defined. In contrast to the bulge ofmurine follicles, which can easily be outlined as a discrete projection, the human adultanagen bulge does not possess distinctive morphological features. Ohyama et al. (2006) firstisolated and characterised stem cell–enriched human hair follicle cells. Some markers havebeen described for these cells, including CD200, K15, and K19. Interestingly, a role has been
proposed for HH signalling in maintaining the human bulge cell phenotype in young andaged human skin (Rittie et al., 2009). Although it has been shown that under severe stressconditions, some bulge “stem” cells can undertake a variety of restorative-related functions,
their role in the undisturbed state is unclear.
(F57) Interfollicular stem cells in human skin. The existence of interfollicular stem cells inhuman skin has been demonstrated, by discriminating three clonal types of keratinocyte withdifferent capacities for multiplication: namely holoclones, meroclones and paraclones(Barrandon and Green, 1987). The entire epithelial compartment of the epidermis of an adult
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human (8 x 1010 cells) can be generated from the progeny of a single stem cell or holoclone.
In cell culture, holoclones are characterised by sustained proliferation, more than 100 PDs
and organogenesis, epidermis reconstruction up to 50 PDs (Fortunel et al., 2010). The
paraclones exclusively contain cells with a short replicative lifespan (<15 PDs), after which
they uniformly abort and terminally differentiate. The meroclone contains a mixture of cellsof different growth potential and is a transitional stage between the holoclone and theparaclone. The concept of the holoclone is still widely used as a criterion for human EpiSCs(Fig. F.3.).

paraclonemeroclone

holoclone
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Fig. F.3. Keratinocyte stem cells are characterised as holoclones (courtesy of MT Martin, CEA,France). (permission needed)(F58) The basal layer of human epidermis is composed of keratinocytes with different
growth potential, as described by Barrandon and Green (1987). The long-term regeneration of
the epidermis is due to the presence of rare cells, which can be isolated as holoclones in tissue
culture.
(F59) The description of membrane markers in keratinocytes with characteristics of stem
cells permitted another classification, according to which the basal layer of epidermis iscomposed of two major cell types, the rare stem cells and their progeny, the keratinocyte
progenitors (Fig. F.4.). Keratinocyte progenitors, also termed TA keratinocytes, constitute themain population of the basal layer. As they divide frequently and give rise to thedifferentiated keratinocytes of the suprabasal layers of the epidermis, they are responsible forthe short-term maintenance of the skin. This population is not homogeneous, ranging fromearly progenitors close to the stem cells up to late progenitors, prepared to migrate into theupper layers, and can be paralleled to the meroclone and paraclone classification.

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Fig. F.4. The classical model of the hierarchical organisation of human epidermis (modified from
Harfouche and Martin, 2010b). (Permission needed)(F60) In homeostatic conditions, KSCs are undifferentiated and slow cycling, which selfrenew through asymmetric division. Their direct progeny, the keratinocyte progenitors, or TA
cells, ensure short-term epidermis maintenance, as they proliferate in the basal compartment
and then migrate to upper layers to give rise to the differentiated keratinocytes.
(F61) Although a variety of membrane markers have been described for KSCs, a uniquemarker is still unavailable and it remains impossible to identify precisely these cells in situ inhuman skin. However, several methods have been described that can select populations
enriched in stem cells from a general population of keratinocytes through cell sorting,including selection on cell size, specific membrane markers, and drug exclusion (Fuchs,
2008; Kaur, 2006; Watt and Jensen, 2009). Small cell size and rapid adhesion to a matrixcoated culture dish have been demonstrated as a way to enrich a keratinocyte population forhuman stem cells from cells freshly isolated from a skin sample (Li et al., 2008). A
population within rapidly adherent keratinocytes that ranged in size from 5–7 µm displayed
the highest density of β1-integrin receptor, contained the highest percentage of cells in G0/G1
phase, showed the highest nucleus to cytoplasm ratio, and possessed the highest colony
forming efficiency. When injected into murine blastocysts, these cells participated in multitissue formation.
(F62) Several membrane markers have been used to enrich cell populations for stem cells
from skin. Combinations of markers based on two KSC properties, quiescence and strong
adhesion to the basal layer of epidermis, are the most frequently used. For adhesion
molecules, the expression of integrins β1 and β6 has been particularly studied. Thus,
enrichment and isolation of a subpopulation of basal epidermal cells from human skin has
been achieved (Li et al., 1998), based on high levels of the adhesion molecule integrin β6 (β6bri), and low levels of the transferrin receptor CD71 (CD71dim). It was shown that cells withthe phenotype β6bri/CD71dim represent the EpiSC population, based on the demonstration thatthese cells exhibit the greatest regenerative capacity of any basal cells, and they represent a
minor subpopulation of basal cells (10%), which are quiescent at the time of isolation fromthe epidermis. Moreover, this strategy was the only one for which serial transplantation

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potential could be demonstrated. The β6bri/CD71dim keratinocytes were able to participate inthree serial epidermis reconstructions in immuno-compromised mice (Terunuma et al., 2007).
Another similar approach used the high expression of adhesion molecules, revealed by a highadhesion capacity, and the low expression of the EGFR (Adh+++EGFRlow phenotype) toisolate KSCs (Fortunel et al., 2003a). These cells exhibited high growth potential in tissue
culture and the long-term capacity to form a pluristratified epidermis.
(F63) A hierarchical lineage structure was also shown by Schluter et al. (2011), using
interfollicular neonatal foreskin epidermis. The tissue was disaggregated, fractionated into
subsets based on markers of keratinocyte stem cells (α6briCD71dim), cycling progenitors(α6briCD71bri), and early differentiating cells (α6dim), followed by functional evaluation using
a limiting dilution in vivo model for tissue reconstitution. The results showed the presence ofa quiescent stem cell population with high long-term epidermal renewal, a cycling progenitor
cell population with rapid but not sustained epidermal reconstitution, and a differentiated cell
population with low regenerative capability.
(F64) One difficult aspect is SC isolation from cultured keratinocytes, because thespecificity of most membrane markers is lost in tissue culture conditions. Cell sorting after
Hoechst labelling has been demonstrated as an efficient method of SC isolation from primary
keratinocyte cultures (Larderet et al., 2006). A family of drug-effluxing pumps, including the
BCRP1/ATP-binding cassette subfamily G member 2 (ABCG2) transporter, allowing theexclusion of DNA dyes such as Hoechst 33342, is more active in stem cells. The small SP ofcells that is able to rapidly exclude the dye, is enriched for stem cells (Redvers et al., 2006).
SP keratinocytes represent 0.16% of the total population, exhibited increased colony-forming
efficiency and long-term expansion potential as compared to other keratinocytes. Importantly,
SP keratinocytes retained the potential to form a pluristratified epidermis even after long-term
culturing.
(F65) Interfollicular stem cells in mouse skin. In mouse skin, the existence and role ofinterfollicular stem cells have long been debated, due to the importance of the follicular stem
cell population. One of the most distinguishing features of stem cells is their slow-cycling
nature. This means that a prolonged period of 3H-thymidine or BrdU staining leads to thelabelling of stem cells, and once labelled, these cells retain the isotope for an extended periodof time during subsequent “chasing”; they can therefore be identified as LRCs. This
technique has been largely used in mice and allowed demonstration that LRCs are found inmouse skin in the hair follicle and interspersed as single cells within the basal layer ofinterfollicular epidermis (Potten and Booth, 2002). These cells perform the long-term
maintenance of epidermis, whereas follicular stem cells are involved in maintenance of thehair follicles.
(F66) Structural considerations of the epidermis on the back of the mouse suggested that the
epidermis is subdivided into a series of functional groupings of cells, each comprising an
individual cell lineage with its own stem cells (Potten, 1986; Potten and Hendry, 1973; Potten
et al., 2002). The cornified layers consist of thin flat cellular elements (squames), which havea large hexagonal surface area, and these are arranged into columns like a stack of plates. The
columns can be traced down to the basal layer within which are a group of about 10 cells withthe responsibility for producing cells for that column, to compensate for the surface squames
that are constantly being lost. As these structures have stability and a long life, they must be
maintained by stem cells. This entire unit was referred to as EPU, and various studies
suggested that each EPU contained a single stem cell situated towards the middle of thecluster of 10 basal cells (Potten, 1986; Potten and Hendry, 1973; Potten et al., 2002). A
similar organisation may exist in human skin, but this has not been clearly demonstrated
because of difficulties in tracing the columns.
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(F67) Follicular stem cells can participate in the reconstitution of damaged epidermis, but
this participation occurs only during wound healing and is transitent. The proof of thedifferent roles of interfollicular and follicular SCs was provided using a model of ablation of
hair follicles in mice. Grafted bulge cells responded to injury by migrating to the wound,participated in the early phases of epidermis regeneration, but were eliminated over several
weeks. Follicular stem cells generated progenitor keratinocytes responsible only for acutewound repair (Ito and Cotsarelis, 2008; Ito et al., 2005). Similarly, mouse stem cells from thehair follicle bulge were found by another group to contribute transiently to interfollicularepidermis wound repair but not to normal homeostasis. In homeostatic conditions, follicular
keratinocytes do not participate in interfollicular renewal (Claudinot et al., 2005).
(F68) The frequency of mouse interfollicular stem cells varies considerably according todifferent authors and is generally thought to be 1 to 10% of the basal cell (Bickenbach et al.,
1986; Heenen and Galand, 1997; MacKenzie, 1985; Morris et al., 1985). A macroscopic,
clonal regeneration assay for mouse epidermis was developed by Withers (Withers, 1967),
which generates nodules very similar in appearance to haematopoietic colonies in the spleenof irradiated mice. Subsequently, Al-Barwari and Potten (1976) developed a microscopic
clonal assay that required a shorter-time interval between irradiation and tissue sampling.
Together, these clonal regeneration assays were interpreted to indicate that only about 10% of
the basal cells have a regenerative capacity, i.e. are stem cells or at least cells capable of somefairly extensive renewal to form colonies. More recently, a functional assay of long-term
repopulating cells found a very low frequency of 1 in 35,000 total epidermal cells, or in theorder of 1 in 104 basal epidermal cells, similar to that of HSCs in the bone marrow (Schneider
et al., 2003).

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F.5. Cellular origin of skin tumours

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(F69) Several models have been proposed concerning skin cell lineages and cancer type.
Sell (2004) speculated that the phenotype of epidermal carcinoma is related to the stage ofdifferentiation of the cell types in the skin where the malignant phenotype is expressed. Thus,in this model, mutated stem cells in the bulge of the hair follicle would give rise totrichoepithelioma, EpiSCs would give rise to BCC, early progenitors would form SCC, andlate progenitor cells would be the origin of papillomas (Fig. F.5.).

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Fig. F.5. Modelling human skin cancer origin (modified from Sell, 2004). (Permission needed)(F70) The principle of the involvement of stem cells and progenitors in humancarcinogenesis is illustrated in the case of UV-induced p53 mutations in human interfollicularepidermis (Owens and Watt, 2003). Numerous cells with p53 mutations can be found in sunexposed, clinically-normal human epidermis. Both scattered single cells and clonal patches ofmutated cells are observed throughout exposed epidermis. The single mutated cells are
predominantly suprabasal, terminally-differentiating cells. The size and distribution of p53mutated patches can be compared with the distribution of stem and progenitors, identified
given that stem cells have relatively higher β1-integrin expression and they tend not to be
actively cycling. Some clones were small and restricted to the progenitor compartment,suggesting that they have arisen from a TA cell founder. Other clones were larger and theirlocation was consistent with a stem-cell founder. Some of these large clones encompassed
several clusters of stem cells. As the location of large patches of p53-mutated cells is
selective for stem-cell-rich regions, the authors proposed that only EpiSCs have the capacity
to substantially propagate UV-induced genetic alterations.
(F71) However, oncogenic events or tumour promoters might also reactivate a stem-cell
programme with self-renewal ability in a target cell without these characteristics (PerezLosada and Balmain, 2003). The initiated ‘de novo stem cell’ might have an increased
adhesion potential: so consequently, it would avoid being discarded by the normaldesquamation process. In fact, skin stem cells express increased levels of adhesion molecules,
such as β1 and β6-integrins, compared with their more differentiated counterparts. This
newly acquired self-renewal capacity could allow these cells to remain within the epidermis
long enough for other oncogenic events, which lead to stabilisation, to accumulate. ‘De novo
stem cells’ could therefore maintain the new tumoural tissue by establishing an alteredhierarchical organisation within the previously normal tissue. The authors thus proposed thatboth stem and progenitor cells may be at the origin of cancer. Concerning the differentiatedkeratinocytes, as none of the benign skin lesions that arose from the suprabasal layers of
epidermis progressed to malignancy, they also stated that reactivation probably does notoccur in these differentiated keratinocytes.

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(F72) Cancer stem cells have not yet been identified in hair follicle or sebaceous tumours.
However, oral SCC initiating tumour cells have been identified and shown to express highlevels of the hyaluronan receptor CD44 (Prince et al., 2007). However, CD44 is not
expressed in human bulge stem cells. It is not known whether a small population of yet-to-beidentified epidermal cells express CD44, or whether de novo expression occurs in SCC
tumour cells. K15 and K19 are highly enriched in hair follicle and sebaceous carcinomas,
suggestive of a bulge origin (Bieniek et al., 2007; Kanitakis et al., 1999). However, as
epithelial progenitors have been described in the sebaceous gland (Lo Celso et al., 2008), theorigin of sebaceous carcinomas could be directly from these glandular cells. SCCs express
high levels of proteins which are normally found in the basal layer of epidermis, including
p63, which is required for proliferation and maintenance of basal keratinocytes (Blanpain andFuchs, 2007; Rocco et al., 2006), and β6-integrin, whose expression is enriched in EpiSCs(Fortunel et al., 2003b). Although expression of stem-cell markers might reflect the tumour
cell of origin, it is only a correlation, and it remains possible that these markers are inducedby the oncogenic events that occur after the initiation of neoplastic growth. In summary,although most data point to a role of stem/progenitor cells, it is not yet demonstrated whichcells are at the origin of human skin cancers.
F.5.1. Cell origin of SCC in mice(F73) Evidence that tumours arise from stem and/or progenitor cells was provided by thetwo-step model of rodent skin carcinogenesis (Sell, 2004). The two steps are initiation andpromotion. In the classic model, DMBA, the initiator, is painted on to the skin. This chemicalbinds to DNA in the skin cells, causing a permanent genetic alteration (initiation) at codon 61
in the Hras gene. However, cancers will not arise unless a proliferative stimulus is also given(promotion). This is provided by treating the skin with 12-O-tetradecanoylphorbor-13-acetate(TPA). Thus, the initiation event induces genetic damage, and the promoter then stimulates
the damaged cells to proliferate, leading to cancer. Initiation must occur before promotion. If
promotion is performed prior to initiation, cancers will not develop. The time between
initiation and promotion is the critical factor in implicating the stem cell as the initiated cell.
This interval can be days, or even months or years in length. In order for tumours to grow inthis model, the initiated cells must survive from the time of initiation to the time of promotion.
Given the well-established fact that all cells in the skin, except for (some of) the stem cells
capable of self-renewal, turn over completely every 2–3 weeks in mice and about 1-2 months
in humans, it is clear that the only way in which the initiated cells could still be present, if
months or years have passed since initiation, would be for initiation to have occurred in a
resting stem cell population. In the course of a year or more between initiation and promotion,
all TA cells would have been replaced by newly generated cells from the basal stem cells.
Thus, in the initiation-promotion model for skin carcinogenesis, the initiated cell must be a
resting stem cell that is only called upon to proliferate under the stress of promotion (Sell,
2004).
(F74) In mouse skin, the hair follicle rather than the interfollicular epidermis is largely
responsible for experimental tumour formation, as most of the mouse skin tumours are SCC
follicle-related (Cotsarelis et al., 1990). As tumour initiation must involve primarily apopulation of long-lived cells, it has been proposed that the follicular stem cells produce
mouse skin tumours. However, targets of tumour initiation can also be found in the mouseinterfollicular epidermis. To determine the origin of skin tumours, the interfollicularepidermis of carcinogen-initiated mice was completely removed by an abrasion techniqueknown to leave the hair follicles undisturbed (Morris et al., 2000). The interfollicular202

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epidermis of the abraded mice quickly regenerated from cells in the hair follicles, after whichtime tumour promotion by chemicals was begun. Mice in which the interfollicular epidermis
had been removed developed papillomas and carcinomas; however, the number of papillomas
was half that of the unabraded mice. These data were consistent with the hypothesis that thetargets of tumour initiation are stem cells found in the hair follicles regarding most malignant
carcinomas, whereas more differentiated cells give rise to papillomas with a low risk ofmalignant conversion.
(F75) Evidence that the consequences of an oncogenic injury depends on the cell thatsustains it, comes from experiments in which the same oncogene is expressed in the hairfollicle or in different layers of the epidermis of transgenic mice (Owens and Watt, 2003;
Preto et al., 2004). When mice were produced with a Ras oncogene which is driven bypromoters that are selectively expressed during terminal differentiation, the only tumours toform were benign papillomas that tended to regress (Bailleul et al., 1990). However, if Ras
was driven by a truncated K5 promoter that was expressed exclusively in the proliferating
cells of the hair follicle, the mice developed malignant carcinomas (Brown et al., 1998).
(F76) Stem cell markers can be studied to address the origin of SCC. In the murine
epidermis, one such candidate marker is CD34, a cell-surface marker that has been used toenrich for multipotent stem cells from the hair follicle bulge (Trempus CS, 2003). Malanchi
et al. (2008) characterised a subpopulation of CD34+ keratinocytes in murine epidermal
tumours induced by chemical (DMBA/TPA) carcinogenesis. The percentage of CD34+
keratinocytes was increased in epidermal tumours (papillomas) and SCCs versus normal
epidermis, although it was not clearly demonstrated whether these cells were progeny ofpreexisting CD34+ cells or de novo CD34-expressing cells. However, the CD34+ population
exhibited phenotypic characteristics of bulge stem cells, including expression of bulge
markers and an absence of the differentiation marker K10. Taken together, these results
indicate that the CD34+ population contains cancer stem cells.
(F77) In chemically induced skin tumours, nuclear β-catenin was enriched in CD34+ versus
CD34- tumour cells (Malanchi et al., 2008), suggesting a potential functional relevance forthis pathway. More importantly, deletion of β-catenin in established tumours led to a
reduction in the percentage of CD34+ cells, and the tumours regressed, as marked byextensive terminal differentiation. The skin tumour regression phenotype was recapitulated
following β-catenin deletion in another mouse skin tumour model (Tg.AC) that expresses
activated H-Ras. Taken together, these data suggest that β-catenin is required for Ras-driven
tumourigenesis in mouse skin, although the mechanism of cancer stem-cell loss following βcatenin deletion is not yet established. However, as noted by the authors, nuclear β-catenin
expression is not strictly a marker of cancer stem cells, and CD34 is not expressed in human
SCC, precluding a stem cell analysis of human tumours. In the human hair follicle, CD34
immunoreactivity is found; however, there is no evidence that this marker enriches for stem
cells. In summary, Malanchi et al. elegantly demonstrated that murine SCCs contain a
subpopulation of cancer stem cells that can be enriched by selection for the follicular bulge
cell surface marker, CD34. They provided convincing evidence that β-catenin signalling isrequired for growth of murine SCC, most likely via the maintenance of stem cells, although
the mechanism by which this occurs is still unclear. Finally, they demonstrated that the
Wnt/β-catenin pathway is also activated in human malignant SCC. However, the challenge
remains to identify cancer stem cells within human epidermal tumours.
(F78) Another group found similar results regarding CD34 and β-catenin. They identified a
population of mouse cells in early epidermal tumours characterised by phenotypic andfunctional similarities to normal bulge skin stem cells. This population contains stem cells,
which are the only cells with tumour initiation properties. Transplants derived from these203

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cells preserve the hierarchical organisation of the primary tumour. They described β-cateninsignalling as being essential in sustaining the cancer stem cell phenotype. Ablation of the βcatenin gene resulted in the loss of stem cells and complete tumour regression. In addition,
they provided evidence for the involvement of increased β-catenin signalling in malignant
human SCCs. Because Wnt/β-catenin signalling is not essential for normal epidermal
homeostasis, such a mechanistic difference may thus be targeted to eliminate cancer stem
cells and consequently eradicate SCCs (Prince and Ailles, 2008). Although these data areinteresting, the data based on markers are questionable, as it has been shown for BCCs thatthey cannot be used to unravel the cellular origin of skin tumours (Youssef et al., 2010).
(F79) In summary, concerning the cellular origin of mouse SCC, transforming events instem cells and/or progenitors are the most frequent early events that lead to SCC formation,although the precise role of each basal keratinocyte population is still to be defined.

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F.6. Radiosensitivity of stem cells and progenitors

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(F82) As skin stem cells self-renew during the whole life of an individual, representing thelong-term reservoir for tissue regeneration, the maintenance of genomic stability should beparticularly stringent in these cells (Harfouche et al., 2010). This maintenance can be realised
through different mechanisms, some specific to stem cells and others which are common withthe differentiated cells.

F.5.2. The cell origin of BCCs
(F80) As in Ptch1neo67/+ mice, skin tumours can originate both from hair follicle andinterfollicular epidermis, and these mice have been used to assess the origin of BCC skintumours. BCC precursor lesions arose from both follicular and interfollicular epithelium in
unirradiated mice, but these lesions progressed to nodular and infiltrative BCCs only in
irradiated mice. Using K14 as a basal differentiation marker, the fact that BCCs originate
from the basal layer of epidermis has been tested. In normal skin, the basal cell layer of theepidermis and the outer root sheath displayed marked immunoreactivity for anti-K14
antibody. No immunoreactivity was detected in the remaining follicular compartments suchas the inner root sheath and the bulb. Basaloid hyperproliferation areas, as well as nodular andinfiltrative BCCs, showed strong immunoreactivity for anti-K14 antibody, suggesting thatthey originate from a common progenitor localised in the basal layer of interfollicularepidermis (Mancuso et al., 2004). The origin of BCC was also addressed by the group ofBlanpain in another model of mice, where they conditionally activate the SHH pathway invarious types of skin cells. Activation was obtained by expressing an active form of the SMO
mutant protein (SmoM2) (see Fig. F.1.). In this model, activation of SmoM2 in hair follicles
did not result in BCC induction. BCC mainly arose without carcinogenic treatment fromlong-term resident progenitors of the interfollicular epidermis, and to a lesser extent fromupper infundibulum (Youssef et al., 2010).
(F81) In summary, the data obtained from two models of mice engineered to modify the
SHH pathway bring interesting findings regarding BCC. In SmoM2- mice, which are modelsof sporadic tumours, stem/progenitors of the interfollicular epidermis were at the origin of
BCCs. In Ptch1+/- mice, which are models for radiation-induced tumours (paragraph F28),
BCCs also appeared to originate from the epidermis. Thus, both models, which may closely
mimick the human BCC pathology, point to the stem/progenitor compartment, without goingin more detail into cell origin.

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(F83) The first mechanism is quiescence, which is possible because a functional hierarchy
exists in epidermis (Fig. F.4.). The most primitive cells, responsible for the long-term renewalof the tissue, divide at a low frequency. Thus, the risk of accumulation of replication errors aswell as fixation of mutations induced by DNA-damaging agents, is reduced in the stem cell
population. The high proliferation rate necessary for turnover of the epidermis is maintainedby the direct progeny of the stem cells, or progenitors, which divide actively. These
progenitors are regularly eliminated by the process of terminal differentiation.
(F84) Another protective mechanism specific to stem cells was proposed by Cairns (1975).
This mechanism is the asymmetrical cell division, giving rise to a stem cell similar to theinitial stem cell, and to another cell type, a progenitor. The group of Fuchs postulated thatstratification occurs through asymmetrical cell divisions in which the mitotic spindle orients
perpendicularly to the basement membrane. They showed in mice that basal epidermal cells
use their polarity to divide asymmetrically, generating a committed suprabasal cell and aproliferative basal cell (Lechler and Fuchs, 2005). Cairns (1975) and then Potten (2004a)
proposed an asymmetrical chromosome segregation resulting in an immortal template DNA
strand remaining in the stem cell and a newly constituted strand being in the progenitor cell.
Such a mechanism would also protect stem cells from replication errors in DNA. To address
this question, the group of Tumbar developed a strategy to count bulge cell divisions inmouse hair follicle and marked them with BrdU. Their study provides quantitative data
supporting the long-standing infrequent SC-division model. However, they showed that hairfollicle stem cells do not retain the older DNA strands or sort their chromosomes (Waghmare
et al., 2008).
(F85) To maintain their genomic stability, stem cells might have developed appropriate
mechanisms of response to DNA-damaging agents. One possible mechanism is theelimination of damaged cells. Cell death, induction of differentiation or senescence of any
damaged stem cells can be a definitive solution to avoid deleterious long-term effects.
Keratinocytes usually die by necrotic processes and mitotic cell death, rather than by
apoptosis, although it is often difficult to distinguish the mode of death in sheet preparationsof epidermis.
(F86) In the mouse, survival data have been published after high doses, based on in vivo
studies. The radiosensitivity of epidermal (macro) colony-forming cells, which possess someof the features of stem cells, was summarised by Potten et al. (1985). From several publisheddata, an average D0 of about 1.2 Gy was determined (Fig. F.6.).

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Fig. F.6. Survival curves for macroscopically visible colonies in mouse skin (Potten et al., 1985). The
number of surviving cells per mm2 of epidermal surface is plotted on a log scale against radiation dose.
The scale on the right shows the surface area needed to provide one surviving cell. The arrow (bottom
right) shows the approximate entire surface area of a mouse. For 3 of the studies, the size of theextrapolation number on the cell survival curve was estimated using split-dose techniques to provide a
recovery factor, or repair capacity (RC). The letters A-G identify curves measured in different studies,
as reviewed by Potten. (Permission needed)(F87) Moreover, the curve for small microscopic colonies, evaluated 3 days after irradiation,
crossed the curves for the larger colonies evaluated at later times. This suggested that mostsmall colonies, produced by the 3 generations of progenitor cells in the epidermis, grew after
irradiation and performed a normal small number of divisions before maturation. This would
be consistent with a relative radioresistance of transit cells. That is in contrast to the highradiosensitivity of transit cells to UV. This difference is also presumed to explain why there
is a longer latency for appearance of skin reactions in the case of x-irradiation compared toafter UV-irradiation (e.g. acute sunburns) (Al-Barwari and Potten, 1979).
(F88) There have been a number of attempts over many years using standard rodent
systems to gather quantitative information on the changes in the fraction of hairs remaining or
surviving (i.e. those not epilated) with varying dose. This should generate a survival curve forfollicular clonogenic cells (stem cells and probably some early progenitor cells). Although
there is some scatter within the data from one set of experiments, each set of experiments
tends to generate a reasonable survival curve. Unfortunately, when comparisons are madebetween sets, the range can be enormous (Hendry et al., 1980; Potten et al., 1985) (Fig. F.7.).
Even with a single strain of mice, where there is considerable genetic uniformity, the results
vary enormously with threshold doses ranging from 4 to 20 Gy and D0 values ranging from1.35 to 6 Gy or even higher. The lower D0 estimates are consistent with the average value forinterfollicular epidermal clonogenic cells. Some of the problems associated with the follicle
survival studies are as follows (Potten et al., 1985):
1. Different endpoints have been used: some workers score variously the hairs shed, thosethat remain, the follicles that remain intact, or the follicles that regrow a new hair. Also,206

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many of these scores have been made at different times after irradiation. Clearly the moststringent test is whether or not a follicle can survive to reorganise a new cycle of hair
growth after irradiation, but this does not necessarily provide the most sensitive survival
curve;
2. The follicles (and the interfollicular epidermis) are subject to various degrees of mild
hypoxia, sufficient to affect radiosensitivity, and this can be modified by environmental
conditions or by the use of anaesthetics;
3. Hair follicles in mice may produce more than one hair, so that follicle and hair counts
may differ;
4. There are different types of hair growing from different-sized follicles which may havedifferent sensitivities. The proportions of the various follicle types may vary from mousestrain to strain and from species to species;
5. Follicles in different stages of the hair growth cycle contain different numbers of cells at
the various stages of the cell cycle;
6. Damage to one follicle may result in the loss of the hair, but the follicle reorganises toregrow another, or alternatively, the follicle may be sterilised and be incapable of further
hair growth. It is conceivable that it may continue to contain a hair for some time eventhough the follicle is sterilised; and7. During the post-irradiation reorganisation of the epithelium, new follicles may be formed
from surviving follicle cells, epidermal cells or sebaceous gland cells. This
reorganisation may take a considerable time.
The range of dose-response curves for hair follicles and hair survival is illustrated in Fig. F.7.
The enormous range in sensitivity is evident. In some cases, anagen hairs are reported to be
more sensitive than telogen hairs, while in other cases, they are said to be more resistant.

Fig. F.7. Survival curves for hairs or hair follicles where the surviving fraction has been plotted on alog scale against the dose on a linear scale (Potten et al., 1985). The labels A-J refer to individual
studies, as reviewed by Potten. (Permission needed)

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F.6.1. Radiation response in vitro(F89) The radiosensitivity of colony-forming primary keratinocytes from childhood
foreskin was measured in vitro (Dover and Potten, 1983). The survival curves showed thatthe cell population was more sensitive (D0 of 0.7-0.9 Gy) than murine epidermal colonyforming cells in vivo (D0 ~ 1.3 Gy). This difference is likely to reflect difference between invivo versus in vitro conditions rather than intrinsic difference in radiosensitivity between
species.
(F90) For human cells, protection from cell death was found in keratinocyte progenitors intissue culture (Tiberio et al., 2002). After isolation from epidermis, cells rapidly adhering tocollagen type IV, with a high level of expression of β1, α6 integrins and p63, were protectedfrom cell death via an integrin signalling pathway in a Bcl-2 dependent manner. Anotherstudy, using the selection of keratinocytes based on the phenotype α6briCD71 (Li et al., 1998),
characterised the response of two cell populations isolated from the basal layer of humanepidermis. The stem cell population (α6briCD71dim), was found radioresistant to γ-irradiation(2 Gy), whereas its direct progeny, the keratinocyte progenitors (α6briCD71bri), was much
more sensitive to the same dose (Rachidi et al., 2007). A 2,3-bis-(2-methoxy-4-nitro-5sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) assay revealed 30% mortality inprogenitors at 72 hours after exposure and none in the stem cells, and colony assays showedthat 82 % of KSCs survived at 2 weeks, as compared to only 29% for the progenitors. A
transcriptome analysis was performed to compare the genomic responses to radiation of thetwo populations. One of the most striking responses of stem cells was the repression of a
network of genes involved in cell death processes. Such a repression can be related to an
important function of stem cells, which is the maintenance of tissue homeostasis, which
requires avoiding massive cell death that would lead to stem cell depletion.
(F91) A major mechanism of resistance to ionising radiation is activation of DNA repair.
In keratinocytes selected with the α6/CD71 phenotype, human KSCs showed more rapid
repair of global DNA damage than in progenitor cells. This result was obtained using thecomet assay, which under alkaline conditions measures mainly single-strand breaks (SSBs).
Moreover, DSBs, measured by the γH2AX assay, were also repaired more rapidly in KSCs
than in progenitor cells. Taken together, these data show that the basal layer of humanepidermis contains two cell populations with different DNA repair capacities, with the minor,
quiescent stem cell population exhibiting a more rapid and efficient repair than the large
progenitor cell population (Harfouche and Martin, 2010).
(F92) Similarly, stem cells from the mouse follicle bulge were found capable of activated
DNA repair capacity. Keratinocytes isolated on the basis of an α6+CD34+ phenotype (hairfollicle bulge cells) were compared to cells with the α6+CD34- phenotype, which represent all
the other keratinocytes, including those of the hair follicle and the basal layer of theepidermis. Although this comparison is poorly selective, it allowed demonstrating that the
repair of both SSBs and DSBs was more rapid in the bulge cells. Moreover, the NHEJ
pathway was found involved in this rapid repair, through DNA-PK activity (Sotiropoulou etal., 2010).
(F93) Finally, activated cell signalling was another possible mechanism of stem cell
resistance. Using the phenotype α6/CD71, activation of the bFGF pathway by DNA damage
was investigated in stem and progenitor cells. The results revealed that the bFGF signalling
pathway was induced by DNA damage in stem cells, but not in progenitor cells. To examine
the role of this endogenous bFGF in DNA repair, stem cells were exposed to bFGF pathway
inhibitors. Blocking the bFGF receptor FGFR1 or the kinase MAPK1 resulted in inhibition of
DNA SSB and DSB repair in the KSCs. Moreover, supplementing the progenitor cells with208

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exogenous bFGF activated their DNA repair. The authors proposed that bFGF helps tomaintain genomic integrity in stem cells by activating stress-induced DNA repair (Harfouche
and Martin, 2010).
(F94) The recent data obtained on human cells and in mice, highlight the importance ofdecrypting the DNA damage response in stem cells. If the increased repair rate observed is an
intrinsic property of EpiSCs, the question of the repair fidelity is crucial concerning thepossible late effects of radiation exposure, such as keratinocyte transformation. In vitrotransformation of normal human keratinocytes is not a common process. Studies on whole
populations of keratinocytes reported that exposing normal cells in tissue culture to various
graded doses of irradiation failed to show any evidence of transformation (Thraves et al.,
1990; Tuynder et al., 1991). When cells were irradiated with 8 fractions of 2 Gy over several
months, and then selected in medium which caused cessation of growth (low EGF
concentration and high Ca2+), one clone escaped senescence but failed to form tumours innude mice (Tuynder et al., 1991).
(F95) Recent studies (Harfouche et al., 2010; Sotiropoulou et al., 2010) point to NHEJ as amechanism of repair for DNA damage in the stem cell population, which can be an errorprone mechanism of repair. On the other hand, keratinocyte progenitors exhibited a slow andincomplete repair, which appears potentially at high risk for cell transformation. In fact, along-term study of genomic instability in cultured clones of progenitors irradiated with 2 Gy
resulted in the occurrence of transformed clones with a surprising high incidence (M Martin,
unpublished data). However, in vivo, the differentiation process should limit this risk, as
keratinocyte progenitors regularly migrate to the upper layers of epidermis to progressively
develop terminal differentiation. An important issue will be to develop cell transformation
assays on purified populations to better characterise the risk for human stem cells and for thedifferent types of keratinocyte progenitors. New models of 3D skin cultures or human grafts
in immune-compromised mice could provide relevant conditions to address this issue.

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F.7. Mutagenesis

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(F96) Mutant Ptch1neo67/+ mice have been used to assess the origin of BCC skin tumours,
as described in section F.3. Also, the origin of BCC was also addressed by the group ofBlanpain in another mouse model, where they conditionally activate the SHH pathway invarious types of skin cells. Activation was obtained by expressing an active form of the
SmoM2 (see section F.3).

8641	
  

F.8. Summary and conclusions

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(F97) The role of KSCs in radiation carcinogenesis is still poorly defined. This is due to
two main reasons. Firstly, knowledge about the biology of EpiSCs is still not sufficient,
illustrated for example by the fact that specific markers of the different cell types found in thebasal layer are lacking. We do not know precisely how to separate stem cells fromprogenitors directly from skin samples, and only enrichment is possible. We do not know
how to characterise the different types of progenitors, from early progenitors close to thestem cells up to the late progenitors committed to migrate to the upper layers of the epidermis.
This is a strong limitation to characterising their role in carcinogenesis.
(F98) Secondly, rodent skin is extremely different from human skin, thus limiting thecomparisons. For example, most mouse tumours are SCCs arising from the stem cells of thebulge, a hair follicle structure. On the contrary, most human epithelial tumours are BCCs
originating from the interfollicular epidermis.

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(F99) Despite these strong limitations, one clear result is that most human epithelial skintumours arise from the basal layer of the epidermis, whereas differentiated keratinocytes giverise to benign lesions such as papillomas. Multiple data argue that EpiSCs can be at the origin
of BCCs, both with and without exposure to ionising radiation. However, some types ofprogenitor cells, still to be defined, also probably participate in carcinogenesis. Concerning
the origin of SCC, there is very little evidence available. In conclusion, the model proposed
by Sell (2004), in which the type of carcinoma depends on the differentiation of the initiallymodified cells, is still a working hypothesis.
(F100) One important question is whether protein markers can be used to elucidate thetarget cells for radiation-induced cancer. Recent data on BCC mouse models demonstrate that
this is probably not the case. Another important question is the management of the radiationstress by the cells. New data, obtained for human and mouse keratinocytes, demonstrate that
more primitive cell populations are able to repair their DNA damage better than their progeny.
Moreover, specific cell signalling upstream of DNA repair can be activated in the stem cells.
It is important to define the type of DNA repair pathways used and the long-term
consequences on genomic stability.
(F101) Trying to answer some of all the remaining questions will require fundamental
studies to better characterise the different types of keratinocytes within the basal layer ofhuman epidermis and the hair follicle. New skin cancer models, such as those derived by thegroup of Khavari, where tumour cells are cultured in 3D environments or after grafting in
immune-compromised mice, will be necessary for improving radiobiological knowledge.
(F102) Finally, the development of cell reprogramming technology, which permits
derivation of embryonic-like stem cells from the skin cells of patients, opens a totally new
area of research. Deriving iPS cell lines from patients with hypersensitive syndromes such as
Gorlin’s syndrome or DC could help better understand the development of radiation-induced
skin tumours.

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ANNEX G: BONE STEM CELLS

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G.1. Radiation carcinogenesis

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(G1) Incorporation of radionuclides into the skeleton has been shown to cause bone cancer
and leukaemia in humans and animals (ICRP, 1991; UNSCEAR, 2000; WHO, 2001).
Radiation-induced tumours are characterised by a latent period, i.e. the time from radiation
exposure to the clinical appearance of a tumour. The latent period consists of two periods: thetrue latent period, or interval from the initiation of cells to the beginning of unrestricted
growth, and a second period of tumour growth, or the time to diagnosis or presentation.
Although the minimum latent period is 3-4 years for bone cancer, the mean latent period forthe induction of bone cancer following external irradiation is 10-15 years (Mettler and Upton,1995).
G.1.1. Radiation-induced bone tumours in humans(G2) An increased incidence of bone tumours has been observed in people exposed to longlived α-emitting isotopes of radium, particularly in painters of luminous dials, but also inradium chemists and in people treated with radium salts in the belief that their effect wastherapeutic (Rundo, 1986). Radium, as an alkaline earth element, behaves similarly tocalcium and is retained predominantly in the skeleton. It is deposited on bone surface andincorporated into bone volume (Leggett et al., 1982). Because of the short range of α particles(40 – 50 µm in soft tissues and less in bone mineral), only atoms decaying on or near to bone
surfaces will irradiate target cells for the induction of bone cancer. In the US, almost 5,000
workers, mainly female, were employed in the luminising industry, mainly between 1915 and1930 and between 1940 and 1954. Fluorescence was achieved initially by addition of 226Ra
salts to paint; later a mixture of 226Ra and 228Ra was also used (Fry, 1998). 226Ra (physicalhalf-life, 1,600 years) emits α particles and decays to 222Rn. 228Ra (half-life 5.75 years)
decays by β particle emission but radioactive progeny include the α particle emitters, 228Th,
224
Ra, 220Rn (called thoron), 216Po, 212Bi and 212Po. By the end of 1983, 62 cases of bonesarcoma had occurred in a total of 2,352 people who had been measured to obtain an estimateof their body content and hence dose (Rundo, 1986). No bone sarcomas were observed atcumulative average bone doses of below 10 Gy (Rowland, 1997).
(G3) 224Ra (half-life 3.6 days) was used in Germany as a treatment for arthritis, ankylosing
spondylitis, and bone tuberculosis in the 1940s and 1950s. The most recent reports of cancer
in patients exposed to high levels of 224Ra (mean cumulative bone surface dose of around 30
Gy, high LET) have included 899 individuals exposed as adults or children (Nekolla et al.,
1999; Nekolla et al., 2000; Nekolla et al., 2005; Spiess, 1995). A total of 56 malignant bonetumours have occurred (0.3 expected), with a peak incidence at 8 years after treatment; only 4
tumours were diagnosed after 1980. Younger ages at exposure, particularly at ages of active
bone growth, appeared to be associated with a higher risk, depending on the dose estimate
used (Henrichs et al., 1995; Nekolla et al., 2000; Nekolla et al., 2005). Lower levels of 224Ra
were given to adult ankylosing spondylitis patients (typical mean cumulative bone surface
dose of about 5 Gy). Follow-up of about 1,500 patients (Wick, 2005; Wick et al., 1999) has
noted small numbers of bone tumours (4 observed, 1.3 expected) and leukaemias (16observed, 6.5 expected).
(G4) Mayak workers were exposed to very high levels of external and internal radiation,
including plutonium exposures in the radiochemical plant and plutonium production plant.

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Levels of irradiation were particularly high during the early years of plant operation (late1940s to the mid-1950s). In the group of 11,000 workers who started work at Mayak during
this time, 27 malignant bone tumours have been observed (19 bone and cartilage neoplasms,
4 myosarcomas, 3 synovial sarcomas and 1 fibrosarcoma) (Koshurnikova et al., 2000). Bonesurface doses were considerably greater than 10 Gy. However, sufficiently reliable estimatesof dose are not yet available, and risk estimates for 239Pu-induced bone cancer have not yet
been made (Harrison and Muirhead, 2003).
(G5) In the past, the terms bone sarcoma or bone cancer, have often been usedsynonymously with osteosarcoma. However, radiation-induced bone sarcomas have beenshown to follow a number of different lines of differentiation. In addition to bone-producing
osteosarcomas, a number of other tumour types have been observed, including fibrosarcomas,
malignant fibrous histiocytoma (MFH) and chondrosarcoma. In two retrospective studies ofbone sarcomas induced by 226,228Ra (Schlenker, 1989) and 224Ra (Gossner et al., 1995), thetwo most common histological types were found to be osteosarcoma (70% for 226,228Ra, 53%
for 224Ra) and non-bone-producing sarcomas of the fibrosarcoma/MFH type (30% for
226,228
Ra, 33% for 224Ra). The high incidence of fibrosarcoma/MFH type tumours, of about
30%, is significantly greater than the incidence of these tumours of about 10% in
spontaneously-occurring bone sarcomas (Gossner, 1999, 2000). It is of interest to note that in
the lower dose 224Ra group referred to above (Wick, 2005; Wick et al., 1999), the small
number of sarcomas observed were not osteosarcomas but included fibrosarcoma/MFH typetumours. The spectrum of radiation-induced bone tumours after exposure to radium isotopes
is similar to that seen after external irradiation (Huvos, 1991; Unni, 1996) and in nonirradiated patients who developed tumours at the site of preexisting bone lesions, such as
Paget’s disease and bone infarct (Desai et al., 1996; Schajowicz, 1993).
(G6) It is possible that gross tissue damage plays an important role in the induction of bonesarcomas by radiation. Radiation is known to cause chronic disturbances of bone remodellingin conditions referred to as osteitis, osteodystrophy, and osteodysplasia. It appears that there
is a low threshold for the induction of such lesions, of about 3 Gy cumulative α skeletal dose
(Hahn et al., 1988). The damage caused is characterised by areas of bone infarction with bone
necrosis, vascular damage and finally peritrabecular fibrosis. A proliferative fibro-osseous
response is also frequently seen in the irradiated marrow. The histopathology of these
responses is similar to the active phase of Paget’s disease, osteitis deformans (Gossner, 1986;
Luz, 1991). Such radiation-induced bone lesions have been described in individuals exposed
to 226Ra. For example, Lisco (1956) reported a fibrosarcoma associated with peritrabecularfibrosis in a radium dial painter. A detailed electron microscope examination of another caseof fibrosarcoma in a radium dial painter by Lloyd and Henning (1983) showed a fibrotic layer
with a thickness of up to 50 µm interposed between marrow cells and the bone surface in theregion of the tumour.
G.1.2. Radiation-induced bone tumours in animals(G7) The toxicity of radium isotopes and other bone-seeking radionuclides has been
compared in beagle dogs in a number of large studies carried out in the US at the Universityof Utah, University of California, Inhalation Toxicology Research Institute and PacificNorthwest Laboratories (Boecker, 1995; Lloyd et al., 2001; WHO, 2001). The results havegenerally been expressed in terms of average bone dose. Comparing bone cancer rates fromlinear dose-response relationships for individual α-emitting nuclides injected systemically insoluble form, toxicity relative to 226Ra was summarised by Boecker et al. (1995) as: 16 for
239
Pu, 9 for 228Th, 6 for 241Am, 4 for 224Ra, and 2 for 228Ra (Boecker, 1995). For the β emitter,
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90

Sr, the dose response was non-linear with no tumours occurring at doses below 18 Gy
cumulative average bone dose. Boecker et al. (1995) quoted values for 90Sr toxicity relative to
226
Ra of 1 ± 0.5 at >40 Gy and 0.05 ± 0.03 at 5 – 40 Gy. The different toxicities of the αemitting nuclides are attributable to differences in dose to the target region near to endostealbone surfaces, which will depend on the affinity of nuclides for different bone surfaces, theirincorporation or burial in bone, and their half-lives. The observed differences between 226Ra
and 90Sr are largely attributable to RBE, with β emissions from 90Sr being substantially lesseffective than α particles except at very high doses.
(G8) Muggenburg et al. (1995) compared the effect of single and multiple administration of224
Ra at levels corresponding to cumulative average bone doses from 0.1–3 Gy (Muggenburg
et al., 1995). Protraction of dose by administration of 224Ra in 50 weekly injections increased
the overall incidence of bone tumours per Gy by a factor of 4. Interpretation of the results iscomplicated by early deaths from marrow dyscrasia in the highest dose, single injectiongroup. Muggenburg et al. (1995) concluded that the incidence per Gy following protracted
administration was very similar to that after injection of 239Pu or inhalation of 238Pu oxide.
(G9) Raabe et al. (1995) analysed data for 226Ra bone cancer induction in dogs in terms of
average skeletal dose rate and time to death. No decrease in lifespan compared to controls
was seen at dose rates of 1 mGy day-1 and less (cumulative doses of <3 Gy) and deaths frommarrow dyscrasia occurred in the high dose region of >100 mGy day-1 (cumulative doses of>100 Gy). At dose rates of 1 – 100 mGy day-1, there was a high incidence of tumours and adecreased latent period with increasing dose rate. Raabe et al. (1995) interpreted these data to
imply that a practical threshold will exist at low dose rates because the time taken for tumour
development will exceed the normal lifespan.
(G10) Osteosarcoma has been observed in mice exposed to 224Ra, 239Pu, 241Am and 233U
(Humphreys et al., 1993; Humphreys et al., 1987; Muller et al., 1990). Ellender et al. (2001)
compared the effect of 239Pu, 241Am and 233U at three levels of activity giving cumulative
average skeletal doses of 0.25–0.3 Gy, 0.5–1 Gy and 1–2 Gy. 233U was considerably lesseffective than 239Pu and 241Am in causing osteosarcoma, consistent with its greater
incorporation into bone mineral due to its chemical similarity to calcium. Osteosarcoma
incidence increased with increasing dose of 239Pu and 241Am, but 239Pu was 2–3 times more
effective per unit average bone dose. Detailed analysis of dose distribution by image analysis
of autoradiographs of bone sections showed that the difference between 239Pu and 241Am inosteosarcoma induction was consistent with their relative delivery of dose to the endosteal
surface (Lord et al., 2001). The best fit between osteosarcoma induction and dose wasobtained by considering dose to a 40-µm layer of marrow adjacent to endosteal surfaces,
although dose to a 10-µm layer also provided a reasonable correlation.
G.1.3. Radiation quality and type of exposure(G11) The 224Ra data discussed above have been used by ICRP to derive a risk estimate forbone tumour mortality of 5 x 10-4 Sv-1, assuming a radiation weighting factor of 20 for α
particles (ICRP, 1991, 2007). This estimate applies to average skeletal dose and would be afactor of 9 lower if estimated on the basis of dose to the bone surface as currently calculated(Eckerman, 1995; Puskin et al., 1992; Spiess, 1995). The data from the follow-up of A-bomb
survivors can be used to provide an estimate of the risk of bone tumours resulting fromexposure to external, mainly low-LET radiation. Using these data, the risk of fatal bone
cancer at low dose and dose rates, applicable to the population of England and Wales, wasestimated as 1 x 10-4 Sv-1 (Muirhead et al., 1993). Grogan et al. (2001) used the analysis of
the A-bomb data by Pierce et al. (1996) to provide an estimate of lifetime risk for the US
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population of around 2–4 x 10-4 Sv-1. The incidence of Paget’s disease is low in Japan,
relative to the US, which may result in underestimation of risk calculated on the basis of Abomb data (Richardson and Cole, 2012). A recent report based on results from the LSS
revealed osteosarcoma to be the most common bone sarcoma, with a dose threshold of 0.85
Gy (Samartzis et al., 2011). These estimates of risk may be regarded as reasonably consistent
with those based on the radium studies, given the uncertainties associated with the A-bombdata and the dose estimates for the 224Ra cases. However, consideration of the risk of bone
cancer based on dose to the bone surface, rather than average bone dose, suggests that the αparticle RBE for bone cancer may be low, although there are substantial uncertainties
associated with each estimate (Harrison and Muirhead, 2003).
(G12) Bone tumours may include types for which there is a threshold dose below which notumours will be observed. It was reported that no sarcomas were observed in 1,339 cases withsystemic intakes of 226Ra/228Ra estimated to give cumulative bone doses of less than 10 Gy;
46 sarcomas were observed in 191 cases with doses greater than 10 Gy (Rowland, 1997;
Rowlands et al., 1995). Analysis of tumour types has shown an increase in radium cases in
the numbers of fibrosarcoma and MFH, relative to osteosarcoma (Gossner, 1999). Tumoursof this type are also known to occur at sites of prexisting non-tumourous bone lesions,
including bone necrosis and fibrous dysplasia, and it may be that the high incidence ofsarcomas of fibrohistiocytic and fibroblastic origin reflects the cell types involved in repair
and remodelling processes. Deterministic tissue damage after high doses of radiation is well
documented in radium-exposed individuals and may be the precursor of fibrosarcoma and
MFH. Another implication of the prevalence of these tumour types is that the target cells may
not be confined to the bone surface lining cells but include cells further into the marrow(Gossner, 2000).
(G13) However, Chadwick et al. (1995) fitted the radium dial painter data using a twomutation carcinogenic model with clonal expansion. The analysis showed that an LQ doseeffect relationship can be applied and, because of the very low natural incidence of bonesarcoma, is consistent with very low risk at low doses and dose rates (Chadwick et al., 1995).

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G.2. General features of bone

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G.2.1. Bone structure(G14) The skeletal system consists of bone, bone marrow, periosteum, all cartilage of thebody, teeth, and the blood vessels and nerves contained in these tissues. Bone consists largelyof an organic matrix impregnated with inorganic salts and permeated by a complex cellular
network (Fig. G.1.). The matrix of bone is composed of various proteins, carbohydrates,
lipids, and other substances, but the bulk of the organic material is made up of a protein
called collagen (Triffitt, 1980). The inorganic matter of bone consists mainly ofsubmicroscopic deposits of forms of calcium phosphate (Fawcett, 1986; Neuman, 1980).

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Fig. G.1. Schematic representation of the lacunar-canalicular system of bone as it opens into thecellular space on the bone surface. From Jaworski (1976). (Permission needed)(G15) Bone develops by transformation of a preexisting connective tissue. Two different
modes of osteogenesis are recognised in embryos. When bone formation occurs directly inprimitive connective tissue, it is known as intramembranous ossification. When it takes placein preexisting cartilage, it is known as intracartilaginous or endochondral ossification(Fawcett, 1986). In endochondral ossification, the bulk of the cartilage must be removed
before bone deposition begins, and neovascularisation of the cartilage anlage has animportant role in the transition from cartilage to bone. The deposition of bone is essentiallythe same in both types of ossification. Bone is laid down first as spongy bone, but some of itis subsequently converted to compact bone by a filling in of the interstices between
trabeculae.
(G16) In bone formation, bone-forming cells (osteoblasts) synthesise the organic matrix, andthis pre-osseous tissue (osteoid) then undergoes mineralisation (Triffitt, 1980). This results ina hard, durable structure which is not permanent. Throughout life, there is a continualmodification (remodelling) of bone by bone resorbing cells, called osteoclasts, and byosteoblasts to maintain the mechanical competence of the structure and to accommodate
conditioning forces that are applied through locomotion, lifting, and the maintenance of
posture (Frost, 1980). Bone remodelling also serves a role in calcium homeostasis (Frost,1980).
(G17) The two main types of bone structure can be distinguished by differences in hardness,porosity, and soft tissue content: compact (cortical) bone and trabecular (cancellous, spongy)
bone. Compact bone is the hard, dense bone that forms the outer wall of all bones, but thebulk of compact bone is found in the shafts of the long bones. Trabecular bone is a soft,
spongy bone composed of a lattice-work of fragile appearance and located at the interior offlat bones and ends of long bones. Trabecular bone has a much higher porosity or soft tissue
content (consisting mainly of bone marrow) and consequently a much lower fractional
volume than compact bone. That is, a much lower portion of volume remains within externalsurfaces of bone after subtraction of volumes of all holes normally occupied by organic
material (Frost, 1963). Not all bone tissue is easily classified as either compact or trabecular,
since there is often a zone between the two bone types that is intermediate in porosity andsurface-to-volume ratio (Parfitt, 1988).

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(G18) Nearly-uniformly-spaced cavities, called lacunae, can be found throughout theinterstitial substance of bone (Fig. G.2.). Each lacuna is filled by a bone cell or osteocyte,
which is essentially an osteoblast that has become surrounded by bone matrix (Fawcett, 1986;
Matthews, 1980). Radiating in all directions from each lacuna is slender, branching tubular
passages, called canaliculi, which penetrate the interstitial substance and join with canaliculi
of neighbouring lacunae. Thus, the lacunae form a continuous system of cavities connectedby an extensive network of minute canals (Fawcett, 1986).

Fig. G.2. Diagram of a sector of the shaft of a long bone illustrating the Haversian systems,
Volkmann’s canals, interstitial lamellae, outer and inner circumferential lamellae, and attachment of
periosteum of bone. From Fawcett (1986). (Permission needed)(G19) The dominant microscopic structure of compact bone is the Haversian system or
osteon (Fig. G.2.). The typical osteon is a cylinder running parallel to the long axis of bone
and is about 200 µm in diameter, but there is considerable variation in the shape, direction,and size of these structures (Fawcett, 1986; Parfitt, 1983). Within the osteon is a central canalabout 40 µm in diameter, containing blood vessels, lymphatics, nerves, and connective tissue.
The walls of the osteon consist of concentric lamellae (layered bone) about 7 µm thick(Parfitt, 1983). Between the Haversian systems of compact bone are irregularly shaped
systems of lamellar bone, called interstitial systems (Fig. G.2.), separated from the Haversian
systems by thin lines of dense connective tissue called cement lines.
(G20) Haversian canals are connected with one another and communicate with the bonemarrow and exterior surfaces of bone via supporting channels called Volkmann’s canals (Fig.
G.2.). Volkmann’s canals are typically oblique or transverse, and are structurally distinct
from Haversian canals in that they are not surrounded by concentrically arranged lamellae but
traverse the lamellae around Haversian systems (Fawcett, 1986). The Haversian systems
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together with Volkmann’s canals, serve to supply nutrients to the canicular network, which inturn carries nutrients to the cells in the interior of compact bone. Cancellous bone has
relatively few Haversian systems and usually consists primarily of angular pieces of lamellarbone (Fawcett, 1986). The bone cells are generally nourished by diffusion from the endosteal
surface via minute canaliculi that interconnect the lacunae and extend to the surface (Fawcett,1986).
(G21) All bones are covered in a fibrous sheath, called the periosteum, except at joint
surfaces. The periosteum consists of a variably thick layer of fibrous connective tissue
consisting of two layers, the outer fibrous layer, and an inner cambium layer (Ellender et al.,1988). The fibrous layer contains fibroblasts and the cambium layer contains progenitor cellswith the potential to develop into bone-forming osteoblasts or cartilage-forming chondrocytes.
In the adult, the progenitors revert to a resting form (Fawcett, 1986), unless activated byevents such as physical damage (e.g. fracture) or novel mechanical stimulation. The
periosteum is penetrated by blood vessels that communicate with Volkmann’s canals, which
in turn communicate with vessels of Haversian canals. The periosteum is abundantly suppliedwith nerves (Moss, 1966). Muscle tendons and ligaments may attach directly into thecompact outer surface of a bone, or they may blend with outer layers of the periosteum (Moss,
1966). The numerous small blood vessels penetrating the periosteum may help keep the
periosteum attached to the underlying bone (Fawcett, 1986). In addition, there are coarse
bundles of collagenous fibres, called Sharpey’s fibres or perforating fibres (Fig. G.2.), thatturn inward from the outer layer of the periosteum and penetrate the outer circumferential
lamellae and intersitital systems of the bone (Fawcett, 1986).
(G22) The endosteum is a layer of cells lining the walls of all cavities in bone that houses
the bone marrow. The endosteum resembles the periosteum in its bone-forming potential but
is much thinner, usually being composed of a single layer of cells without associatedconnective tissue fibres. All cavities of bone, including the Haversian canals and the marrow
spaces within trabecular bone, are lined by endosteum (Fawcett, 1986).
(G23) In the adult human, the typical long bone is composed of a central cylindrical shaft
called a diaphysis, two roughly spherical, terminal articular regions, called epiphyses and twointermediate cone-like regions, called metaphyses, that connect the shaft and articular ends.
In growing children, the epiphysis is separated from the diaphysis by a cartilaginous
epiphyseal plate, which is united to the diaphysis by columns of trabecular bone in themetaphysis.
(G24) The flat bones of the skull generally lack a central marrow region, but consist of twoplates of compact bone with an intervening trabecular region, called the diploë (Fawcett,
1986; Moss, 1966). The outer surfaces of both plates are covered with a periosteum, and the
diploic space is lined with an endosteum. In the case of the bones of the skull vault, the outersurface is lined by a connective tissue covering called the pericranium, and the inner surface
is lined by the dura mater of the brain; these linings do not differ greatly in structure orfunction from the periosteum and endosteum of the long bones (Fawcett, 1986; Moss, 1966).
G.2.2. Cell types and location(G25) Precursors of bone tumours must be cells with proliferative potential, and thus bothstem cells and pluripotent progenitor cells are candidate precursors for radiation-inducedbone tumours described above (Basu-Roy et al., 2012a). In the osteoblastic lineage, mature
osteocytes and osteoblasts are non-proliferative cells. Preosteoblasts are dividing cells
committed to osteoblast formation with a limited proliferation capacity (Dorfman and217

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Czerniak, 1998). Osteoprogenitor cells, precursor of the preosteoblast, have a higher
proliferation capacity (Gossner, 2003).
(G26) Stem cells for the osteoblast lineage reside in the bone marrow and other soft tissues.
Populations of marrow-derived stem cells that are capable of osteogenesis were first
identified by Friedenstein et al. (1970) who showed that when plated at clonal density, a
subpopulation of plastic-adherent, bone marrow-derived cells formed colonies (CFU-F) andcould differentiate to form bone and cartilage tissue after heterotopic transplantation(Friedenstein et al., 1987). Over the years, pluripotent progenitors and stem cells withosteogenic potential were assigned various names and identified by diverse biologicalattributes in part due to population heterogeneity with respect to self-renewal. In 2005, newcriteria and nomenclature for defining mesenchymal stromal/stem cells (MSCs) were
proposed by the International Society for Cellular Therapy, and accepted widely in the field(Dominici et al., 2006; Horwitz et al., 2005; Keating, 2012). The minimum criteria fordefining MSC (which refers to both mesenchymal stromal cells and mesenchymal stem cells)
include adherence to plastic, pluripotent differentiation (adipogenic, chondrogenic andosteogenic cells) and expression of select cell-surface markers (see Section G.2.5); not allcells within a population of MSC retain the capacity to self-renew (Dominici et al., 2006;
Horwitz et al., 2005; Keating, 2012). Although this is currently an active area of research,
there remains uncertainty regarding self-renewal (‘stemness’) and pluripotency of MSC in
situ (Bianco et al., 2008; Keating, 2012). Once transplanted heterotopically or grown underselect culture conditions, both human and murine bone marrow-derived MSC, retain thepotential to replicate and differentiate into different bone cell types, including chondrocytes,
adipocytes, osteoblasts and fibroblasts (Pittenger et al., 1999). Circulating MSC also may
contribute to tissue repair, including at sites of bone fracture and radiation damage (Khosla etal., 2010; Mouiseddine et al., 2007). Further, a subpopulation of circulatory stem cells withosteogenic potential for tissue repair may derive from a common HSC (Pignolo and Kassem,2011), although further study is needed regarding lineage.
(G27) The bone marrow adjacent to bone surfaces is a complex tissue containing aconnective tissue network, known as stroma. It consists of fibroblast-like stromal cells, MSCs,
macrophages, and quiescent CD34- HSCs. Developments in stem cell biology indicate that
there is a high degree of plasticity in the stem cell pool (Gossner, 2003; Huss, 2000). It has
been shown that CD34- cells, as well as generating CD34+ haematopoietic progenitors, canalso regenerate committed mesenchymal precursors, such as osteoblasts, chondrocytes andmyocytes. Thus, it appears that CD34- stem cells as well as mesenchymal precursors are
possible target cells for radiation-induced bone cancer.
(G28) Bone surfaces include both quiescent areas covered by thin layers of inactive
osteoblasts, referred to as lining cells (Eriksen, 2010) and active, formative surfaces coveredby contiguous osteoblasts. These are recognised histologically by eccentric nuclei andbasophilic cytoplasm, due to a high content of rough endoplasmic reticulum engaged in
production of the extracellular matrix (osteoid). The ratio of these two surfaces will varydepending on the extent of ongoing bone remodelling required under normal steady-state
conditions or as a consequence of radiation-associated pathology (Seed et al., 1982). Since
preosteoblasts, their committed osteoprogenitor precursors, MSCs and CD34- stem cells are
not normally located directly in contact with bone surfaces, the current assumption (ICRP,
1975) of a cell layer thickness as low as 10 µm being the target for radiation-induced bone
cancer does not appear to be appropriate.
(G29) Gössner (2003) considered that in general terms, the targets for radiation
tumourigenesis may not be confined to the stem cells and committed progenitor cells inwhich DNA mutation may result in transformation as a step towards malignancy. Such cells
218

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exist in a microenvironment of surrounding normal cells, vasculature and extracellular matrix,
each of which may become involved in the tumourigenic process. This view is in accord within vitro observations of non-targeted (bystander) effects in which damage responses such as
apoptosis and genomic instability are seen in cells that have not been directly irradiated,presumably by cell–cell signalling (Prise et al., 2002). This is an area of active experimental
research, but current evidence for bystander effects occurring in vivo is limited and their rolein tumourigenesis remains to be determined (Brooks, 2004; Kassis, 2004; Morgan and Bair,2013).
G.2.3. Tissue turnover rate(G30) As discussed above, bone has three distinct cell types: osteoblasts, or bone-forming
cells, osteoclasts, or bone-resorbing cells and osteocytes, which are terminally differentiatedosteoblasts entrapped within lacunae. Osteoblasts create and maintain skeletal architecture
and are responsible for the deposition of bone matrix and for the regulation of osteoclasts.
Osteoblasts are mononuclear, not terminally differentiated, specialised cells (Canhao et al.,2005) and form tight junctions with adjacent osteoblasts and regions of plasma membrane
specialised in vesicular trafficking and secretion (Mackie, 2003). As they differentiate, theyacquire the ability to secrete bone matrix (Gori et al., 2000). The vast majority of osteoblasts(60-90%) undergo apoptosis rather than become osteocytes or inactive lining cells (Jilka et al.,2007; Parfitt, 1990). Ultimately, some osteoblasts become trapped in their own bone matrix,
giving rise to osteocytes, which gradually stop secreting osteoid (Mackie, 2003). Osteocytes
are the most abundant cells in bone and communicate with each other and with thesurrounding medium through extensions of their plasma membrane (Knothe Tate et al., 2004;
Manolagas, 2000). Therefore, osteocytes are considered to act as mechanosensors, instructing
osteoclasts where and when to resorb bone and osteoblasts, also where and when to form it(Manolagas, 2000; Nakashima et al., 2011; Seeman and Delmas, 2006).
(G31) Osteocytes can undergo apoptosis, and remain entrapped within the mineralised
matrix. Apoptosis of osteocytes is thought to provide signals for resorption of bone in
response to localised or humoral stimuli (Aguirre et al., 2006; Tomkinson et al., 1997).
Although high dose radiation reduces osteocyte density and induces bone resorption(Sugimoto et al., 1993; Takahashi et al., 1994), animal studies suggest that osteocyte
apoptosis does not account directly for acute, radiation-mediated bone resorption (Midgley etal., 1995).
(G32) In order to balance bone formation and resorption in healthy individuals, osteoblasts
secrete factors that regulate the differentiation of osteoclasts, and osteocytes secrete factors
regulating the activity of both osteoblasts (Hartmann, 2006) and osteoclasts (Seeman andDelmas, 2006). Bone is constantly remodelled in a dynamic system where osteoblasts are
responsible for bone formation and osteoclasts for its resorption (Ducy et al., 2000). The net
amount of bone formed is a function of the number and/or the activity of mature osteoblasts.
Targeted ablation of 70-80% of osteocytes leads to cortical porosity and bone loss (Tatsumi
et al., 2007). Thus, both proliferation of cells within the osteoblast lineage and death ofosteocytes may each contribute to bone formation or the regulation of bone resorption,although neither is likely to be required for remodelling to proceed. Resorption is much fasterthan formation: an area of bone can be resorbed in 2-3 weeks but it takes at least 3 months torebuild (Harada and Rodan, 2003). The typical remodelling cycle lasts 6-9 months in healthy
adult humans and relies on the generation of new osteoblasts and osteoclasts. Therefore,
remodelling is dependent throughout life on a continuous supply of stem cells andprogenitors to replenish the pools of precursors depleted during remodelling.
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G.2.4. Age and gender specificity of tissue turnover(G33) The number of MSCs in the body decreases with age (Fibbe and Noort, 2003) andinfirmity (Inoue et al., 1997). The greatest numbers of MSCs are found in neonates andnumbers reduce throughout life to about half at the age of 80 (Fibbe and Noort, 2003). In thefetus, the highest number of circulating MSCs is detected during the first trimester anddeclines during the second trimester to about 0.0001 % and further to 0.00003 % of nucleatedcells in cord blood (Campagnoli et al., 2001). It has been suggested that uncommitted MSCs
circulate during gestation and travel from fetal sites into other tissues during early
development (Makino et al., 1999).
(G34) As mentioned previously, bone sarcomas are most prevalent in the young relative toold adults, peaking in the 10-19 year age group with a second peak arising at a later age (>65
years) (Bleyer et al., 2006; Ottaviani and Jaffe, 2009). There is also a gender difference;
young males are more frequently affected than young females, though mortality rates arecomparable. One possible explanation for an increased tumour incidence in 10-19 years old isthat puberty corresponds to a period of rapid skeletal growth and skeletal modelling,
rendering conditions favourable to transformation and tumourigenesis, analogous to thesensitivity of sites associated with Paget’s lesions.
G.2.5. Cellular features(G35) MSCs isolated as plastic-adherent multipotent cells, capable of differentiating into
bone, cartilage and fat cells, can be isolated from many tissues as well as from bone marrow(Davis and Zur Neiden, 2008; Kolf et al., 2007). They express a range of specific surface
antigens, but a definitive marker for MSCs has yet to be identified, hampering studies of celllineage and niche location. The most widely used MSC markers are CD90, CD73 and CD105(Dominici et al., 2006). Stro-1 has been identified as an MSC marker and Stro-1- cell
populations are not capable of forming colonies (i.e. not containing CFU-F) (Simmons and
Torok-Storb, 1991). Stro-1+ cells can become HSC supporting fibroblasts, smooth muscle
cells, adipocytes, osteoblasts and chondrocytes (Dennis and Charbord, 2002). However, Stro1 is unlikely to be a general MSC marker since its expression is not exclusive to MSCs
(Gronthos et al., 2003). Its mouse counterpart has not been known and its function remains tobe elucidated. CD106 or vascular cell adhesion molecule 1 (VCAM-1) is expressed on bloodvessel endothelial and adjacent cells, consistent with a perivascular location of MSCs. It is
likely to be functional in MSCs, being involved in cell adhesion, chemotaxis and signaltransduction (Carter and Wicks, 2001). CD106 singles out about 1% of Stro-1+ cells, which
are the only Stro-1+ cells that form colonies and show stem cell characteristics such as
multipotentiality, expression of telomerase, and high proliferation in vitro (Gronthos et al.,2003). Notably, primary human osteosarcomas and chondrosarcoma cells express key
transcription factors characteristic of MSC and their differentiated progeny, including Sox9(chondrocytes), runt-related transcription factor 2 (Runx2) (osteoblasts), Sox2 and MET(osteosarcoma) (Basu-Roy et al., 2012b; Dani et al., 2012; Tang et al., 2010; Wagner et al.,2011). Further, cloned cell lines from osteosarcomas or chondrosarcomas selected for CD133
continue to express stem cell makers, and retain both tumourigenicity and the ability todifferentiate into osteoblasts and adipocytes (Grosse-Gehling et al., 2013; Martins-Neves etal., 2012; Tirino et al., 2011).
(G36) A number of factors have been implicated in the maintenance of MSCs as stem cells,
including leukaemia inhibitory factor (LIF) (Jiang et al., 2002; Metcalf, 2003), FGFs
220

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(Tsutsumi et al., 2001; Zaragosi et al., 2006), and Wnt (Boland et al., 2004; Kleber andSommer, 2004). LIF, a pleiotropic cytokine, maintains the stem cell state of MSCs and otherstem cells, and also regulates osteoblast/osteoclast activity. The MSC niche may be locatedperivascularly as indicated by the expression of α-smooth muscle actin in MSCs isolated
from all tissue types tested (Meirelles, 2006) and the immunohistochemical localisation of
CD45-/CD31-/Sca-1+/Thy-1+ cells to such sites (Blashki et al., 2006). MSCs identified by
Stro-1 and CD146 markers have been located to blood vessel linings in bone marrow anddental pulp (Shi, 2003). Localisation of MSCs to perivascular niches is consistent with theirhaving ready access to and a role in repair of damage in many different tissues. The capacityfor MSCs to home to different tissues seems to be related in part to expression of Stro-1(Bensidhoum et al., 2004). Whereas Stro-1- cells were better able to aid in the engraftment
and survival of HSCs, Stro-1+ cells were more capable of homing and engrafting to mosttissues studied. In vitro studies show that MSC migration is regulated by stromal-derived
factor-1/CXCR4 and hepatocyte growth factor/c-Met complexes, and involves matrix
metalloproteinases (Son et al., 2006). In vivo studies have shown that injury alters thepatterns of migration and differentiation of exogenously added sites but also promoted
widespread engraftment to multiple organs (Francois et al., 2006).
(G37) The Wnt signalling pathway has been shown to play a vital role in the process ofdifferentiation of osteoblasts, chondrocytes and adipocytes from stem and progenitor cells invivo and in vitro (Davis and Zur Neiden, 2008). The Wnt family of ligands consists of anumber of highly evolutionarily conserved secreted glycoproteins involved in many
developmental processes, such as cell proliferation, differentiation, polarity, migration andregeneration (Huelsken J., 2002; Moon RT, 2002). Wnts are essential for normal
embryogenesis and also control tissue-specific stem cell activity postnatally in the intestine,skin, and other tissues. The vertebrate Wnts have been divided into two functional groups: thecanonical and non-canonical pathways. In the canonical pathway, Wnt molecules attach tothe membrane-bound receptor Frizzled (Fzd) and to a coreceptor called low-density
lipoprotein receptor related protein 5/6 (LRP5/6) (Cadigan and Liu, 2006; Krishnan et al.,2006). In the absence of a Wnt ligand, a multiprotein complex involving axin, casein kinase 1,
glycogen synthase kinase 3β, APC, and Dishevelled (Dsh) mediate the degradation of βcatenin. Wnt signalling allows the accumulation of β-catenin which enters the nucleus andbinds to the transcription factors lymphoid enhancore factor (LEF) and/or T-cell factor (TCF),
triggering downstream gene transcription, including that of c-myc and Cyclin D1 (Logan and
Nusse, 2004). The non-canonical Wnts are less well characterised, but do not signal throughβ-catenin, and in some cases, may inhibit nuclear β-catenin activity (Ishitani et al., 2003). The
canonical and non-canonical pathways are not distinct, as some Wnts can signal through both
pathways (Kuhl et al., 2000) and some downstream targets are involved in both, such as Dsh(Yang Y, 2003). The action of the pathway members appears to depend on the cellularcontact. In stem cells, Wnt/β-catenin signalling regulates cellular function and maintains“stemness”. However, both the canonical (nuclear β-catenin activity) and non-canonicalcascade (blockage of nuclear β-catenin activity) also control differentiation.
(G38) As well as controlling self-renewal and early differentiation, the Wnt signalling
pathway thereafter specifically regulates the lineage-specification of mesenchymal precursor
cells into adipocytes, osteoblasts and chondrocytes (Ross et al., 2000; Sommer, 2002; Tosh
and Slack, 2002). However, not only biochemical signals, but also physical conditions suchas cell density and cell shape appear to play a role in lineage commitment. Mesenchymal
condensations are characterised by increased cell density and cell-cell adhesion. Lower celldensities appear to support osteoblast differentiation whereas higher cell densities cause cells
to condense, forcing differentiation into adipocytes (McBeath R, 2004). Cell shape may be
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regulated by RhoA, a downstream target of the non-canonical Wnt pathway. Cell-celladhesion involves N-cadherin which directly interacts with β-catenin at the cell membrane,
specifically at the time of mesenchymal condensation prior to differentiation (Tuan, 2003). βcatenin activity is essential for the differentiation of mature osteoblasts and bone formation.
Lack of β-catenin does not change the differentiation of osteoprogenitor cells into the early
osteoblastic precursors, but blocks the expression of transcription factor osterix (Osx), andconsequently, the cells acquire a chondrogenic phenotype (Hartmann, 2006).
(G39) During differentiation, osteoblasts express a characteristic pattern of genes that
distinguish them from other cell types (Caetano-Lopes et al., 2007). Collagen type Iα1(COL1) is expressed from the beginning of osteoblast differentiation and is the main
structural component of bone matrix. Alkaline phosphatase (ALP) is upregulated early duringosteoblast differentiation, and both osteopontin (OPN) and ALP are important in stabilising
the matrix. Osteocalcin is another non-collagenous protein, which is almost exclusively
expressed in bone and is upregulated in the late differentiation stage. This stage coincides
with the onset of mineralisation, suggesting that osteocalcin may play a part in the regulationof bone matrix mineralisation (Thomas et al., 2001).
(G40) Osteoblasts have been shown to have a role in the regulation of bone resorption
through receptor activator of nuclease factor-κB (RANK) ligand (RANKL) that links to its
receptor, RANK, on the surface of preosteoclast cells, inducing their differentiation andfusion. Osteoblasts also secrete a soluble decoy receptor (osteoprotegerin, OPG) that blocks
RANK/RANKL interaction by binding to RANKL, and thus prevents osteoclastdifferentiation and activation, reducing bone resorption (Krane, 2005). Therefore, the balance
between RANKL and OPG determines the formation and activity of osteoclasts (Gori et al.,2000).

9197	
  G.3. Radiosensitivity

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(G41) MSCs are considered relatively radioresistant. Long-standing evidence forradioresistance of the stromal compartment is based on the findings that bone stromal cells
and MSCs are typically of host origin following bone marrow ablation (Bartsch et al., 2009;
Rieger et al., 2005). The relative radioresistance of MSCs is attributed to the fact that thecells are quiescent or divide very slowly in situ (Morikawa et al., 2009). Even when
maintained in vitro in the presence of serum growth factors that stimulate proliferation,
relatively high doses of radiation are required to trigger apoptosis and inhibit CFU in human
MSCs (hMSCs) [D0 = 2 Gy for hMSCs obtained from a commercial source (Chen et al.,2006)].
(G42) Cultures of cells obtained from bone marrow and capable of forming colonies whenplated at clonal density include a subpopulation that is pluripotent and capable ofosteogenesis, although there is not a one-to-one correlation between CFU-F and CFUosteoblast (CFU-Ob; osteogenic cells) (Morikawa et al., 2009). Therefore, earlier lessons
learned regarding radiosensitivity and genomic instability from experiments performed onstromal cells that were simply flushed from the marrow and seeded on to plastic, also are
pertinent to what are now described as MSCs., e.g. (Epperly et al., 1999; Friedenstein et al.,
1981; Greenberger et al., 1982). The D0 for radiation-induced inhibition of colony formation(CFU-F) from guinea pig marrow is reported to be 2 Gy (Friedenstein et al., 1981) just as for
hMSCs (Chen et al., 2006)). Substantial differences in cell culture conditions, purification
methods and immunophenotyping and characterisation between experiments contribute
substantial uncertainty to quantitative values reported in the literature, and this remains asignificant challenge in this field.

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(G43) With respect to MSCs, radiosensitivity can be considered in the context ofdifferentiation, pluripotency and senescence, in addition to the more traditional survival
assays for apoptosis and colony formation. Radiation-induced changes in differentiation
potential are particularly relevant, considering the mounting evidence that stem cell niches
and the marrow microenvironment produced by various differentiated progeny of MSCs,
contribute to carcinogenesis. When interpreting results from cell culture experiments on MSC
growth and differentiation, it is important to take into account the fact that the vast majorityof studies do not employ clonal plating densities: therefore, observed changes in cell
behaviour and molecular pathways typically reflect the behaviour of a heterogeneous
population of cells that are not clonal in origin.
G.3.1. Characteristics of damage response at the cell level(G44) Irradiation can adversely affect various key cell functions in MSCs, including
proliferation, differentiation, apoptosis, senescence and perhaps pluripotency. Recent
evidence demonstrates that mouse MSCs express high levels of various DNA damage
response proteins (ATM, Chek2, and DNA Ligase IV), high levels of anti-apoptotic proteins
and low levels of pro-apoptotic proteins, which together are likely to confer relative
radioresistance of MSCs compared to other cell types (Sugrue et al., 2013a, b). Even after
high dose irradiation, there is evidence that MSCs retain the capacity for osteogenic
differentiation, albeit at reduced levels [e.g. 9 Gy in vitro irradiation of hMSC (Chen et al.,2006) and 7Gy of mouse MSC (Mehrara et al., 2010)]. Osteoblast differentiation in culture ishighly density-dependent: therefore, in cases where irradiation inhibits differentiation, theresponses actually may arise due to insufficient cell density. Nonetheless, a radiation-induced
decrease in bone formation by osteoblasts has potential adverse consequences for bothmineralised tissue and bone marrow.
(G45) The influence of radiation on MSC growth, differentiation and senescence in cell
culture depends to a large extent on the stage of cell growth at the time of exposure, withconfluent cells showing modest effects on osteoblast differentiation markers (Ikeda et al.,
2000; Kurpinski et al., 2009; Wang and Jang, 2009). Irradiation of hMSCs with x-rays or 56Fe
(1 Gy) at 70% confluence, does not impair osteogenic differentiation, but does reduce growthand induce senescence (Kurpinski et al., 2008; Wang and Jang, 2009). In contrast, primary
hMSCs irradiated in suspension, then plated to grow in osteogenic or adipogenic media,
display reduced osteogenic differentiation and proliferation (Li et al., 2007). As in humancells, mouse bone marrow stromal cells immunophenotyped for MSC markers exposed tohigh doses (>7 Gy) during exponential growth showed reduced proliferation and osteogenic
differentiation with increased markers for apoptosis (Mussano et al., 2010).
(G46) Animal studies support cell culture results showing that irradiation can impair theosteogenic differentiation in marrow progenitors and stem cells. In mice, total body
irradiation (TBI) with 7 Gy of low-LET x-rays or 0.5 Gy or high-LET 56Fe ions reduces
subsequent ex vivo osteoblastogenesis from bone marrow cells (Mehrara et al., 2010; Yumoto
et al., 2010). Irradiation of mice (4 Gy 137Cs, TBI) reduces both the numbers and osteoblast
differentiation of MSCs ex vivo, immunoselected using an endothelial progenitor cell marker,
vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) (Ma et al., 2007). A radiationinduced reduction in ex vivo osteogenesis is also associated with a reduction in mass(osteopenia) of rapid turnover, trabecular bone, although the acute loss of bone is most likely
due to increased bone resorption by osteoclasts; radiation-induced deficits in bone formationby progeny of MSC are most likely to influence bone structure only at later times during
subsequent bone turnover (Kondo et al., 2009; Willey et al., 2008; Yumoto et al., 2010). Thus,
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results from animal studies support findings from the post-surgical clinical application ofionising radiation to prevent heterotopic ossification (Balboni et al., 2006), perhaps in part,by impairing MSC responses to local growth factors that are released as a consequence oftissue injury (Pohl et al., 2003).
(G47) Ionising radiation may also influence the differentiation fate of MSCs and pluripotent
progenitors. In humans, radiogenic marrow ablation leads to increased marrow adiposity(Rosen et al., 2009). One hypothesis to explain these and related findings is that adipocytes
differentiate at the expense of osteoblasts, due to a ‘switch’ in differentiation at the level of ashared progenitor cell (Calo et al., 2010). Differences in the dose dependence of radiationinduced changes in adipogenic versus osteogenic differentiation markers expressed bymesenchymal progenitors are reported in some but not all studies (Li et al., 2007; Schonmeyr
et al., 2008).
(G48) In summary, the preponderance of evidence indicates that ionising radiation can
influence MSC growth, senescence and differentiation, although further study is needed todetermine if radiation can influence differentiation fate (Mehrara et al., 2010; Schonmeyr etal., 2008).
(G49) Irradiation of MSCs, progenitors, and pluripotent cell lines stimulates key
radiosensitive signalling and transcriptional pathways including Rb, p53/p21, ATM and
MAPK (Chen et al., 2006; Jin et al., 2008; Kurpinski et al., 2009; Liang et al., 2011; Wangand Jang, 2009). Radiation exposure can also influence cell behaviour by modulatingcytokine signalling pathways. Irradiation of the pluripotent cell line C2C12 during
exponential growth interferes with BMP2-receptor complex formation and inhibits
osteogenic differentiation (Pohl et al., 2003), but has little effect on differentiation if cells arenear confluence at the time of irradiation (Ikeda et al., 2000). In addition, treatment with theneuropeptide, Substance P, prolongs MAPK activation and mitigates radiation-induced
apoptosis of hMSC in vitro and bone marrow-derived CFU-F in vivo (An et al., 2011).
(G50) As mentioned above, MSC and HSC reside in situ within a low oxygen-tension
environment, which may protect cells from radiation damage. MSC appear to possess robust
antioxidant ROS-scavenging capacity and DNA repair mechanisms, which may contribute totheir relative radioresistance (Chen et al., 2006; Sugrue et al., 2013b).

9299	
  

G.4. Mutagenesis

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(G51) Specific radiation-induced gene mutations leading to transformation in MSC have notbeen identified to date, although there is limited cell culture evidence that irradiation of
hMSCs can lead to malignant transformation. hMSCs transduced to express elevatedtelomerase levels did not actively restore their chromosome-arm specific telomere-length
pattern after exposure to ionising radiation (Graakjaer et al., 2007). Further, irradiation of atelomerase-transduced human mesenchymal stem cell line led to chromosomal instability andtumour formation in SCID mice (Christensen et al., 2008; Horwitz et al., 2005; Keating,2012; Martins-Neves et al., 2012; Sugrue et al., 2013a; Sugrue et al., 2013b). These results
argue that in principle, irradiation of bone marrow-derived MSCs can directly cause
transformation.

9310	
  

G.5. Summary

9311	
  
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• Incorporation of radionuclides into the skeleton causes bone cancer and leukaemia inhumans and animals.

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• Radiation-induced bone sarcomas include osteosarcomas, fibrosarcomas, MHF andchondrosarcoma.
• Tissue damage may play an important role in the induction of bone sarcomas byradiation, as radiation causes chronic disturbances of bone remodelling in conditions
referred to as osteitis, osteodystrophy and osteodysplasia.
• Based on 224Ra data, the risk estimate for bone tumour mortality is 5 x 10-4 Sv-1,
assuming a radiation weighting factor of 20 for α particles (applied to an average skeletal
dose) (ICRP, 1991, 2007). For estimates of the risk for bone tumours resulting fromexposure to external, mainly low-LET radiation, the A-bomb survivor data provide anestimate of 1 x 10-4 Sv-1 for the population of England and Wales and a lifetime risk
around 2–4 x 10-4 Sv-1 for the US population.
• MSCs are identified as plastic-adherent pluripotent cells, capable of differentiating into
bone, cartilage and fat cells, and can be isolated from many different tissues in addition
to bone marrow. MSCs express high levels of DNA damage repair proteins, arerelatively radioresistant (D0 ~2 Gy), and possess robust antioxidant ROS-scavenging
capacity.
• Exposure to ionising radiation can adversely affect various key cell functions of cultured
MSCs (proliferation, differentiation, senescence) and also can cause loss of bone massand skeletal fragility in both animals and humans.
• MSCs and quiescent CD34- HSCs are candidate precursors for radiation-induced bonetumours.
• MSCs and primitive HSCs reside in situ within a low oxygen-tension environment,
which may help protect cells from radiation damage.

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REFERENCES
Abelin, T., Egger, M., Ruchti, C., 1994. Fallout from Chernobyl. Belarus increase was probably
caused by Chernobyl. Br. Med. J. (Clin Res Edition) 309, 1298-1300.
Abramson D.H., K.F.D., Tung G., Ellsworth R.M., 1984. Second nonocular tumors in retinoblastoma
survivors. Are they radiation-induced? Opthalmology 91, 1351-1355.
Adams, L.M., Ethier, S.P., Ullrich, R.L., 1987. Enhanced in vitro proliferation and in vivo
tumorigenic potential of mammary epithelium from BALB/c mice exposed in vivo to gammaradiation and/or 7,12-dimethylbenz[a]anthracene. Cancer Res. 47, 4425-4431.
Adiga, S.K., Toyoshima, M., Shiraishi, K., et al., 2007. p21 provides stage specific DNA damage
control to preimplantation embryos. Oncogene 26, 6141-6149.
Aguirre, J.I., Plotkin, L.I., Stewart, S.A., et al., 2006. Osteocyte apoptosis is induced byweightlessness in mice and precedes osteoclast recruitment and bone loss. J. Bone Miner. Res.
21, 605-615.
Al-Barwari, S.E., Potten, C.S., 1976. Regeneration and dose-response characteristics of irradiatedmouse dorsal epidermal cells. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 30, 201-216.
Aladjem, M.I., Spike, B.T., Rodewald, L.W., et al., 1998. ES cells do not activate p53 dependentstress responses and undergo p53 independent apoptosis in response to DNA damage. Curr.
Biol. 8, 145-155.
Alberg, A.J., Brock, M.V., Samet, J.M., 2005. Epidemiology of lung cancer: looking to the future. J.
Clin. Oncol. 23, 3175-3185.
Albert, R.E., Burns, F.J., Heimbach, R.D., 1967. The effect of penetration depth of electron radiation
on skin tumor formation in the rat. Radiat. Res. 30, 515-524.
Allen, R.D.r., Bender, T.P., Siu, G., 1999. c-Myb is essential for early T cell development. Genes Dev.
13, 1073-1078.
Allen, T.D., Dexter, T.M., 1976. Cellular interrelationships during in vitro granulopoiesis.
Differentiation; Res. in Biol. Div. 6, 191-194.
Allred, D.C., Medina, D., 2008. The relevance of mouse models to understanding the development
and progression of human breast cancer. J. Mammary Gland Biol. Neoplasia 13, 279-288.
Allsopp, R.C., Cheshier, S., Weissman, I.L., 2001. Telomere shortening accompanies increased cell
cycle activity during serial transplantation of hematopoietic stem cells. J. Exp. Med. 193, 917924.
Alvi, A.J., Clayton, H., Joshi, C., et al., 2003. Functional and molecular characterisation of mammaryside population cells. Breast Cancer Res. 5, R1-8.
Amos C.I., X.W., Spitz M.R., 1999. Is there a genetic basis for lung cancer susceptibility? Recent
Results Cancer Research 151, 3-12.
An, Y.S., Lee, E., Kang, M.H., et al., 2011. Substance P stimulates the recovery of bone marrow after
the irradiation. J. Cell Physiol. 226, 1204-1213.
Andarawewa, K.L., Erickson, A.C., Chou, W.S., et al., 2007. Ionizing radiation predisposesnonmalignant human mammary epithelial cells to undergo transforming growth factor beta
induced epithelial to mesenchymal transition. Cancer Res. 67, 8662-8670.
Andersson, M., Carstensen, B., Visfeldt, J., 1993. Leukemia and other related hematological disorders
among Danish patients exposed to Thorotrast. Radiat. Res. 134, 224-233.
Anisimov, V.N., Ukraintseva, S.V., Yashin, A.I., 2005. Cancer in rodents: Does it tell us about cancer
in humans? Nat. Rev. Cancer. 5, 807-819.
Antonchuk, J., Sauvageau, G., Humphries, R.K., 2001. HOXB4 overexpression mediates very rapid
stem cell regeneration and competitive hematopoietic repopulation. Exp. Hematol. 29, 11251134.
Arai, F., Hirao, A., Ohmura, M., et al., 2004. Tie2/angiopoietin-1 signaling regulates hematopoietic
stem cell quiescence in the bone marrow niche. Cell 118, 149-161.
Archer, V.E., Wagoner, J.K., Lundin, F.E., 1973. Lung cancer among uranium miners in the UnitedStates. Health Phys. 25, 351-371.

226

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9391	
  
9392	
  
9393	
  
9394	
  
9395	
  
9396	
  
9397	
  
9398	
  
9399	
  
9400	
  
9401	
  
9402	
  
9403	
  
9404	
  
9405	
  
9406	
  
9407	
  
9408	
  
9409	
  
9410	
  
9411	
  
9412	
  
9413	
  
9414	
  
9415	
  
9416	
  
9417	
  
9418	
  
9419	
  
9420	
  
9421	
  
9422	
  
9423	
  
9424	
  
9425	
  
9426	
  
9427	
  
9428	
  
9429	
  
9430	
  
9431	
  
9432	
  
9433	
  
9434	
  
9435	
  
9436	
  
9437	
  
9438	
  
9439	
  
9440	
  
9441	
  
9442	
  
9443	
  

Ariazi, J.L., Haag, J.D., Lindstrom, M.J., et al., 2005. Mammary glands of sexually immature rats are
more susceptible than those of mature rats to the carcinogenic, lethal, and mutagenic effects ofN-nitroso-N-methylurea. Molecular Carcinogenesis 43, 155-164.
Armakolas, A., Klar, A.J., 2006. Cell type regulates selective segregation of mouse chromosome 7
DNA strands in mitosis. Science 311, 1146-1149.
Armitage, P., Doll, R., 1954. The age distribution of cancer and a multi-stage theory of carcinogenesis.
Br. J. Cancer 8, 1-12.
Artandi, S.E., DePinho, R.A., 2010. Telomeres and telomerase in cancer. Carcinogenesis 31, 9-18.
Artavanis-Tsakonas, S., Rand, M.D., Lake, R.J., 1999. Notch signaling: cell fate control and signal
integration in development. Science 284, 770-776.
Arufe, M.C., Lu, M., Kubo, A., et al., 2006. Directed differentiation of mouse embryonic stem cells
into thyroid follicular cells. Endocrinology 147, 3007-3015.
Arvold, N.D., Taghian, A.G., Niemierko, A., et al., 2011. Age, breast cancer subtype approximation,and local recurrence after breast-conserving therapy. Journal of Clinical Oncology 29, 38853891.
Asaithamby, A., Hu, B., Delgado, O., et al., 2011. Irreparable complex DNA double-strand breaks
induce chromosome breakage in organotypic three-dimensional human lung epithelial cell
culture. Nucleic Acids Research 39, 5474-5488.
Atwood, C.S., Hovey, R.C., Glover, J.P., et al., 2000. Progesterone induces side-branching of theductal epithelium in the mammary glands of peripubertal mice. J. Endocrinol. 167, 39-52.
Awad, Z.T., Watson, P., Filipi, C.J., et al., 1999. Correlations between esophageal diseases andmanometric length: a study of 617 patients. J. Gastrointestinal Surgery 3, 483-488.
Bachoo, R.M., Maher, E.A., Ligon, K.L., et al., 2002. Epidermal growth factor receptor andInk4a/Arf: convergent mechanisms governing terminal differentiation and transformation along
the neural stem cell to astrocyte axis. Cancer cell 1, 269-277.
Bailey-Wilson, J.E., Amos, C.I., Pinney, S.M., et al., 2004. A major lung cancer susceptibility locus
maps to chromosome 6q23-25. Am. J. Hum. Genet. 75, 460-474.
Bailleul, B., Surani, M.A., White, S., et al., 1990. Skin hyperkeratosis and papilloma formation intransgenic mice expressing a ras oncogene from a suprabasal keratin promoter. Cell 62, 697708.
Bair, W.J., Dagle, G.E., James, A.C., 1989. Overview of biological consequences of exposure toplutonium and higher actinides. Radiation Protect. Dosim. 26, 125-135.
Balboni, T.A., Gobezie, R., Mamon, H.J., 2006. Heterotopic ossification: Pathophysiology, clinicalfeatures, and the role of radiotherapy for prophylaxis. Int. J. Radiat. Oncol. Biol. Phys. 65,
1289-1299.
Banerjee, M.R., Chakraborty, S., Kinder, D., et al., 1987. Cell biology of mouse mammary
carcinogenesis in organ culture. In: Medina, D., Kidwell, W., Heppner, G., Anderson, E. (Eds.),
Cellular and Molecular Biology of Mammary Cancer. Plenum Press, New York, pp. 353-380.
Bao, S., Wu, Q., McLendon, R.E., et al., 2006. Glioma stem cells promote radioresistance bypreferential activation of the DNA damage response. Nature 444, 756-760.
Barbera, M., di Pietro, M., Walker, E., et al., 2014. The human squamous oesophagus has widespread
capacity for clonal expansion from cells at diverse stages of differentiation. Gut. doi:
10.1136/gutjnl-2013-306171. [Epub ahead of print]
Barcellos-Hoff, M.H., 1993. Radiation-induced transforming growth factor b and subsequent
extracellular matrix reorganization in murine mammary gland. Cancer Res. 53, 3880-3886.
Barcellos-Hoff, M.H., 1994. A novel redox mechanism for TGF-beta activation. Mol. Biol. Cell. 5,
139a.
Barcellos-Hoff, M.H., 1998. The potential influence of radiation-induced microenvironments inneoplastic progression. J. Mammary Gland Biol. Neoplasia 3, 165-175.
Barcellos-Hoff, M.H., Ravani, S.A., 2000. Irradiated mammary gland stroma promotes the expressionof tumorigenic potential by unirradiated epithelial cells. Cancer Res. 60, 1254-1260.
Barker, N., Ridgway, R.A., van Es, J.H., et al., 2009. Crypt stem cells as the cells-of-origin of
intestinal cancer. Nature 457, 608-611.

227

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9444	
  
9445	
  
9446	
  
9447	
  
9448	
  
9449	
  
9450	
  
9451	
  
9452	
  
9453	
  
9454	
  
9455	
  
9456	
  
9457	
  
9458	
  
9459	
  
9460	
  
9461	
  
9462	
  
9463	
  
9464	
  
9465	
  
9466	
  
9467	
  
9468	
  
9469	
  
9470	
  
9471	
  
9472	
  
9473	
  
9474	
  
9475	
  
9476	
  
9477	
  
9478	
  
9479	
  
9480	
  
9481	
  
9482	
  
9483	
  
9484	
  
9485	
  
9486	
  
9487	
  
9488	
  
9489	
  
9490	
  
9491	
  
9492	
  
9493	
  
9494	
  
9495	
  
9496	
  

Barker, N., van de Wetering, M., Clevers, H., 2008. The intestinal stem cell. Genes & development 22,
1856-1864.
Barker, N., van Es, J.H., Kuipers, J., et al., 2007. Identification of stem cells in small intestine andcolon by marker gene Lgr5. Nature 449, 1003-1007.
Barrandon, Y., Green, H., 1987. Three clonal types of keratinocyte with different capacities for
multiplication. Proc. Natl. Acad. Sci. U.S.A. 84, 2302-2306.
Barrocal, V., 2009. Mouse differentiating spermatogonia can generate germinal stem cells in vivo.
Nature Cell Biology, 190-196.
Bartel, F.O., Higuchi, T., Spyropoulos, D.D., 2000. Mouse models in the study of the Ets family oftranscription factors. Oncogene 19, 6443-6454.
Bartkova, J., Horejsi, Z., Koed, K., et al., 2005. DNA damage response as a candidate anti-cancer
barrier in early human tumorigenesis. Nature 434, 864-870.
Bartsch, H., Hollstein, M., Mustonen, R., et al., 1995. Screening for putative radon-specific p53
mutation hotspot in German uranium miners. Lancet 346, 121.
Bartsch, K., Al-Ali, H., Reinhardt, A., et al., 2009. Mesenchymal stem cells remain host-derived
independent of the source of the stem-cell graft and conditioning regimen used. Transplantation
87, 217-221.
Basu-Roy, U., Basilico, C., Mansukhani, A., 2013. Perspectives on cancer stem cells in osteosarcoma.
Cancer Lett. 338, 158-167.
Basu-Roy, U., Seo, E., Ramanathapuram, L., et al., 2012. Sox2 maintains self renewal of tumorinitiating cells in osteosarcomas. Oncogene 31, 2270-2282.
Bauer, F.W., Crombag, N.H., de Grood, R.M., et al., 1980. Flow cytometry as a tool for the study ofcell kinetics in epidermis. Br. J. Dermatol. 102, 629-639.
Bauer, F.W., Grood, R.M., 1975. Impulse cytophotometry in psoriasis. Br. J. Dermatol. 93, 225-227.
Baverstock, K., Egloff, B., Pinchera, A., et al., 1992. Thyroid cancer after Chernobyl. Nature 359, 2122.
Baxter, N.N., Tepper, J.E., Durham, S.B., et al., 2005. Increased risk of rectal cancer after prostateradiation: a population-based study. Gastroenterology 128, 819-824.
Becker, D., Elsasser, T., Tonn, T., et al., 2009. Response of human hematopoietic stem and progenitor
cells to energetic carbon ions. Int. J. Radiat. Biol. 85, 1051-1059.
Beckwith, J.B., Perrin, E.V., 1963. In situ neuroblastomas: a contribution to the natural history ofneural crest tumors. Am. J. Pathol. 43, 1089-1104.
BEIR_V, 1990. Health Effects of Exposure to Low Levels of Ionizing Radiation:BEIR V. The
National Academies Press, USA.
BEIR_VI, 1999. Health Effects of Exposure to Radon:BEIR VI. The National Academies Press, USA.
BEIR_VII, 2006. Health Risks from Exposure to Low Levels of Ionizing Radiation: BEIR VII Phase
2. The National Academies Press, USA.
Belinsky, S.A., Klinge, D.M., Liechty, K.C., et al., 2004. Plutonium targets the p16 gene forinactivation by promoter hypermethylation in human lung adenocarcinoma. Carcinogenesis 25,
1063-1067.
Belinsky, S.A., Middleton, S.K., Picksley, S.M., et al., 1996. Analysis of the K-ras and p53 pathways
in X-ray-induced lung tumors in the rat. Radiat Res. 145, 449-456.
Bensidhoum, M., Chapel, A., Francois, S., et al., 2004. Homing of in vitro expanded Stro-1- or Stro1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting
human CD34 cell engraftment. Blood 103, 3313-3319.
Bergmann, O., Bhardwaj, R.D., Bernard, S., et al., 2009. Evidence for cardiomyocyte renewal in
humans. Science 324, 98-102.
Bergstresser, P.R., Chapman, S.L., 1980. Maturation of normal human epidermis without an orderedstructure. Br. J. Dermatol. 102, 641-648.
Bertoncello, I., Williams, B., 2004. Hematopoietic stem cell characterization by Hoechst 33342 andrhodamine 123 staining. Methods in Mol. Biol. 263, 181-200.
Bhardwaj, G., Murdoch, B., Wu, D., et al., 2001. Sonic hedgehog induces the proliferation ofprimitive human hematopoietic cells via BMP regulation. Nat. Immunol. 2, 172-180.

228

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9497	
  
9498	
  
9499	
  
9500	
  
9501	
  
9502	
  
9503	
  
9504	
  
9505	
  
9506	
  
9507	
  
9508	
  
9509	
  
9510	
  
9511	
  
9512	
  
9513	
  
9514	
  
9515	
  
9516	
  
9517	
  
9518	
  
9519	
  
9520	
  
9521	
  
9522	
  
9523	
  
9524	
  
9525	
  
9526	
  
9527	
  
9528	
  
9529	
  
9530	
  
9531	
  
9532	
  
9533	
  
9534	
  
9535	
  
9536	
  
9537	
  
9538	
  
9539	
  
9540	
  
9541	
  
9542	
  
9543	
  
9544	
  
9545	
  
9546	
  
9547	
  

Bianco, P., Robey, P.G., Simmons, P.J., 2008. Mesenchymal stem cells: revisiting history, concepts,
and assays. Cell Stem Cell, 2, 313-319.
Bickenbach, J.R., McCutecheon, J., Mackenzie, I.C., 1986. Rate of loss of tritiated thymidine label inbasal cells in mouse epithelial tissues. Cell Tissue Kinet. 19, 325-333.
Bieniek, R., Lazar, A.J., Photopoulos, C., et al., 2007. Sebaceous tumours contain a subpopulation ofcells expressing the keratin 15 stem cell marker. Br. J. Dermatol. 156, 378-380.
Bissell, D.M., 2001. Chronic liver injury, TGF-beta, and cancer. Exp. Mol. Med. 33, 179-190.
Bissell, M.J., Aggeler, J., 1987. Dynamic reciprocity: How do extracellular matrix and hormones
direct gene expression? In: Cabot, M. (Ed.), Mechanisms of Signal Transduction by Hormones
and Growth Factors. Alan R. Liss, Inc., New York, pp. 251-262.
Bithell, J.F., Stiller, C.A., 1988 A new calculation of the carcinogenic risk of obstetric X-raying. Stat
Med. 7, 857-864.
Blair, C., 1968. Morphology and thickness of the human stratum corneum. Br. J. Dermatol. 80, 430436.
Blanpain, C., Fuchs, E., 2007. p63: revving up epithelial stem-cell potential. Nat. Cell. Biol. 9, 731733.
Blanpain, C., Lowry, W.E., Geoghegan, A., et al., 2004. Self-renewal, multipotency, and the existenceof two cell populations within an epithelial stem cell niche. Cell 118, 635-648.
Blasco, M.A., 2007. Telomere length, stem cells and aging. Nature Chem. Biol. 3, 640-649.
Blashki, D., Short, B., Bertoncello, I., et al., 2006. Identification of stromal MSC candidates frommultiple adult mouse tissues. Int. Soc. Stem Cell Res. 4th Annual Meeting, 206.
Bleyer, A., O'Learn, M.R.B., et al., 2006. Cancer epidemiology in older adolescents and young adults
15 to 29 years of age. In: Ries, L.A.G., Smith, M.A., Gurney, L.G., Linet, M., Tamra, T.,
Young, J.L., Bunin, G.R. (Eds.), Cancer Incidence and Survival among Children andAdolescents: United States SEER program 1975-1995. National Cancer Institute, Bethesda,
MD., USA.
Boecker, G., Guilmette, R.A., Hahn, F.F., et. al., 1995. The role of laboratory animals in the studying
the late-occuring effects of radionuclides deposited in the liver and skeleton. In: Health Effects
of Internally Deposited Radionuclides: Emphasis on Radium and Thorium. World Scientific,
Singapore, pp. 287-297.
Boers, J.E., Ambergen, A.W., Thunnissen, F.B., 1999. Number and proliferation of clara cells innormal human airway epithelium. Am. J. Respir. Crit. Care Med. 159, 1585-1591.
Boice, J.D., Jr., Blettner, M., Kleinerman, R.A., et al., 1987. Radiation dose and leukemia risk inpatients treated for cancer of the cervix. J. Nat. Can. Inst. 79, 1295-1311.
Boice, J.D., Jr., Day, N.E., Andersen, A., et al., 1985. Second cancers following radiation treatment
for cervical cancer. An international collaboration among cancer registries. J. Nat. Can. Inst. 74,
955-975.
Boice, J.D., Jr., Engholm, G., Kleinerman, R.A., et al., 1988. Radiation dose and second cancer risk inpatients treated for cancer of the cervix. Radiat. Res. 116, 3-55.
Boice, J.D., Jr., Fry, R.J., 1995. Radiation carcinogenesis in the gut, in: Potten, C.S., Hendry, J.H.
(Eds.), Radiation and Gut. Elsevier, pp. 291-306.
Boice, J.D., Jr., Miller, R.W., 1999. Childhood and adult cancer after intrauterine exposure to ionizing
radiation. Teratology 59, 227-233.
Boice Jr., J.D., 2001. Radiation and breast carcinogenesis. Med. Pediat. Onc. 36, 508-513.
Boice Jr., J.D., Harvey, E.B., Blettner, M., et al., 1992. Cancer in the contralateral breast afterradiotherapy for breast cancer. N. Engl. J. Med. 326, 781-785.
Boice Jr., J.D., Preston, D., Davis, F.G., et al., 1991. Frequent chest x-ray fluoroscopy and breast
cancer incidence among tuberculosis patients in Massachusetts. Radiat. Res. 125, 214-222.
Boland, C.R., Sato, J., Appelman, H.D., et al., 1995. Microallelotyping defines the sequence andtempo of allelic losses at tumour suppressor gene loci during colorectal cancer progression. Nat.
Med., 902-909.

229

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9548	
  
9549	
  
9550	
  
9551	
  
9552	
  
9553	
  
9554	
  
9555	
  
9556	
  
9557	
  
9558	
  
9559	
  
9560	
  
9561	
  
9562	
  
9563	
  
9564	
  
9565	
  
9566	
  
9567	
  
9568	
  
9569	
  
9570	
  
9571	
  
9572	
  
9573	
  
9574	
  
9575	
  
9576	
  
9577	
  
9578	
  
9579	
  
9580	
  
9581	
  
9582	
  
9583	
  
9584	
  
9585	
  
9586	
  
9587	
  
9588	
  
9589	
  
9590	
  
9591	
  
9592	
  
9593	
  
9594	
  
9595	
  
9596	
  
9597	
  
9598	
  
9599	
  
9600	
  

Boland, G.M., Perkins, G., Hall, D.J., et al., 2004. Wnt 3a promotes proliferation and suppresses
osteogenic differentiation of adult human mesenchymal stem cells. J. Cell Biochem. 93, 12101230.
Bommer, G.T., Gerin, I., Feng, Y., et al., 2007. p53-mediated activation of miRNA34 candidate
tumor-suppressor genes. Curr. Biol. 17, 1298-1307.
Bond, V.P., 1995. Dose, effect severity, and imparted energy in assessing biological effects. StemCells 13, 21-29.
Bondar, T., Medzhitov, R., 2010. p53-mediated hematopoietic stem and progenitor cell competition.
Cell Stem Cell 6, 309-322.
Booth, D., Haley, J.D., Bruskin, A.M., et al., 2000. Transforming growth factor-β3 protects murine
small intestinal crypt stem cells and animal survival after irradiation, possibly by reducing
stem-cell cycling. Int. J. Cancer 86, 53-59.
Booth, D., Potten, C.S., 2001. Protection against mucosal injury by growth factors and cytokines. J.
Natl. Cancer Inst., 16-20.
Borthwick, D.W., Shahbazian, M., Krantz, Q.T., et al., 2001. Evidence for stem-cell niches in the
tracheal epithelium. Am. J. Respir. Cell Mol. Biol. 24, 662-670.
Bouffler, S.D., Breckon, G., Cox, R., 1996. Chromosomal mechanisms in murine radiation acute
myeloid leukaemogenesis. Carcinogenesis 17, 655-659.
Bounacer, A., Wicker, R., Caillou, B., et al., 1997. High prevalence of activating ret proto-oncogene
rearrangements, in thyroid tumors from patients who had received external radiation. Oncogene
15, 1263-1273.
Bouras, T., Pal, B., Vaillant, F., et al., 2008. Notch Signaling Regulates Mammary Stem Cell Function
and Luminal Cell-Fate Commitment. Cell Stem Cell 3, 429-441.
Bowie, M.B., McKnight, K.D., Kent, D.G., et al., 2006. Hematopoietic stem cells proliferate until
after birth and show a reversible phase-specific engraftment defect. J. Clin. Invest. 116, 28082816.
Boyd, S.D., 2008. Everything you wanted to know about small RNA but were afraid to ask. Lab.
Invest. 88, 569-578.
Brambilla, E., Gazzeri, S., Lantuejoul, S., et al., 1998. p53 mutant immunophenotype andderegulation of p53 transcription pathway (Bcl2, Bax, and Waf1) in precursor bronchial lesionsof lung cancer. Clin. Cancer. Res. 4, 1609-1618.
Brambilla, E., Moro, D., Gazzeri, S., et al., 1999. Alterations of expression of Rb, p16(INK4A) andcyclin D1 in non-small cell lung carcinoma and their clinical significance. J. Pathol. 188, 351360.
Brambilla, E., Negoescu, A., Gazzeri, S., et al., 1996. Apoptosis-related factors p53, Bcl2, and Bax inneuroendocrine lung tumors. Am. J. Pathol. 149, 1941-1952.
Brellier, F., Valin, A., Chevallier-Lagente, O., et al., 2008. Ultraviolet responses of Gorlin syndrome
primary skin cells. Br. J. Dermatol. 159, 445-452.
Brennan, P., Crispo, A., Zaridze, D., et al., 2006. High cumulative risk of lung cancer death amongsmokers and nonsmokers in Central and Eastern Europe. Am. J. Epidemiol. 164, 1233-1241.
Bresciani, F., 1968. Topography of DNA synthesis in the mammary gland of the C3H mouse and its
control by ovarian hormones: an autoradiographic study. Cell. Tiss. Kinet. 1, 51-63.
Broeks, A., Braaf, L.M., Wessels, L.F., et al., 2010. Radiation-associated breast tumors display a
distinct gene expression profile. Int. J. Radiat. Oncol. Biol. Phys. 76, 540-547.
Brooks, A.L., 2004. Evidence for 'bystander effects' in vivo. Human & Exp. Toxicol. 23, 67-70.
Brown, K., Strathdee, D., Bryson, S., et al., 1998. The malignant capacity of skin tumours induced byexpression of a mutant H-ras transgene depends on the cell type targeted. Current Biology 8,
516-524.
Brownson, R.C., Alavanja, M.C., Caporaso, N., et al., 1998. Epidemiology and prevention of lungcancer in nonsmokers. Epidemiol Rev. 20, 218-236.
Bruenger, F.W., Lloyd, R.D., Miller, S.C., 1991. The influence of age at time of exposure to 226Ra or
239
Pu on distribution, retention, postinjection survival, and tumor induction in beagle dogs.
Radiat. Res. 125, 248-256.

230

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9601	
  
9602	
  
9603	
  
9604	
  
9605	
  
9606	
  
9607	
  
9608	
  
9609	
  
9610	
  
9611	
  
9612	
  
9613	
  
9614	
  
9615	
  
9616	
  
9617	
  
9618	
  
9619	
  
9620	
  
9621	
  
9622	
  
9623	
  
9624	
  
9625	
  
9626	
  
9627	
  
9628	
  
9629	
  
9630	
  
9631	
  
9632	
  
9633	
  
9634	
  
9635	
  
9636	
  
9637	
  
9638	
  
9639	
  
9640	
  
9641	
  
9642	
  
9643	
  
9644	
  
9645	
  
9646	
  
9647	
  
9648	
  
9649	
  
9650	
  
9651	
  
9652	
  
9653	
  

Brugmans, L., Kanaar, R., Essers, J., 2007. Analysis of DNA double-strand break repair pathways inmice. Mut. Res. 614, 95-108.
Brüske-Hohlfeld, I., Rosario, A.S., Wölke, G., et al., 2006. Lung cancer risk among former uranium
miners of the Wismut Company in Germany. Health Physics 90, 208-216.
Burns, F.J., Albert, R.E., 1986a. Dose-response for radiation-induced cancer in rat skin. In: Burns, F.J.,
Upton, A.C., Silini, G. (Eds.), Radiation Carcinogenesis and DNA Alterations. Plenum Press,
New York, pp. 51-70.
Burns, F.J., Albert, R.E., 1986b. Radiation carcinogenesis in rat skin. In: Radiation Carcinogenesis.
Elsevier, New York, Amsterdam, London, pp. 199-214.
Burns, F.J., Albert, R.E., Sinclair, I.P., et al., 1975. The effect of 24-hour fractionation interval on the
induction of rat skin tumors by electron radiation. Radiat. Res. 62, 478-487.
Burns, F.J., Sinclair, I.P., Albert, R.E., et al., 1976. Tumor induction and hair follicle damage fordifferent electron penetrations in rat skin. Radiat. Res. 67, 474-481.
Burns, F.J., Vanderlaan, M., 1977. Split-dose recovery for radiation-induced tumours in rat skin. Int. J.
Radiat. Biol. 32, 135-144.
Burstein, D.E., Nagi, C., Wang, B.Y., et al., 2004. Immunohistochemical detection of p53 homolog
p63 in solid cell nests, papillary thyroid carcinoma, and hashimoto's thyroiditis: A stem cellhypothesis of papillary carcinoma oncogenesis. Human Pathol. 35, 465-473.
Buske, C., Feuring-Buske, M., Abramovich, C., et al., 2002. Deregulated expression of HOXB4
enhances the primitive growth activity of human hematopoietic cells. Blood 100, 862-868.
Caddle, L.B., Hasham, M.G., Schott, W.H., et al., 2008. Homologous recombination is necessary fornormal lymphocyte development. Mol. Cell. Biol. 28, 2295-2303.
Cadigan, K.M., Liu, Y.I., 2006. Wnt signaling: complexity at the surface. J. Cell. Sci. 119, 395-402.
Caetano-Lopes, J., Canhao, H., Fonseca, J.E., 2007. Osteoblasts and bone formation. Acta ReumatolPort 32, 103-110.
Cai, W.B., Roberts, S.A., Potten, C.S., 1997. The number of clonogenic cells in crypts in three regionsof murine large intestine. Int. J. Radiat. Biol. 71, 573-579.
Cairns, J., 1975. Mutation selection and the natural history of cancer. Nature 255, 197-200.
Calin, G.A., Sevignani, C., Dumitru, C.D., et al., 2004. Human microRNA genes are frequently
located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. U S A.
101, 2999-3004.
Calo, E., Quintero-Estades, J.A., Danielian, P.S., et al., 2010. Rb regulates fate choice and lineage
commitment in vivo. Nature 466, 1110-1114.
Calvi, L.M., Adams, G.B., Weibrecht, K.W., et al., 2003. Osteoblastic cells regulate thehaematopoietic stem cell niche. Nature 425, 841-846.
Cameselle-Teijeiro, J., Varela-Duran, J., Sambade, C., et al., 1994. Solid cell nests of the thyroid:
light microscopy and immunohistochemical profile. Human Pathol. 25, 684-693.
Campagnoli, C., Roberts, I.A., Kumar, S., et al., 2001. Identification of mesenchymal stem/progenitor
cells in human first-trimester fetal blood, liver, and bone marrow. Blood 98, 2396-2402.
Campbell, F., Williams, G.T., Appleton, M.A., et al., 1996. Post-irradiation somatic mutation andclonal stabilisation time in the human colon. Gut 39, 569-573.
Canhao, H., Fonseca, J.E., Viana Querioz, M., 2005. Mechanismos de remodelcao ossea e factoresprotectors do osso. Acta Reumatol. Port. 30, 225-240.
Cardis, E., Vrijheid, M., Blettner, M., et al., 2007. The 15-country collaborative study of cancer risk
among radiation workers in the nuclear industry: estimates of radiation-related cancer risks.
Radiat. Res. 167, 396-416.
Carpenter, M.K., Rosler, E., Rao, M.S., 2003. Characterization and differentiation of humanembryonic stem cells. Cloning and Stem cells 5, 79-88.
Carr, Z.A., Kleinerman, R.A., Stovall, M., et al., 2002. Malignant neoplasms after radiation therapy
for peptic ulcer. Radiat. Res. 157, 668-677.
Carsten, A.L., 1984. Acute Lethality - The hemopoietic syndrome in different species. In: Broerse, J.J.,
Macvittie, T.J. (Eds.), Response of Different Species to Total Body Irradiation. Martinus
Nijhoff Publishers, Dordrecht, pp. 59-86.

231

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9654	
  
9655	
  
9656	
  
9657	
  
9658	
  
9659	
  
9660	
  
9661	
  
9662	
  
9663	
  
9664	
  
9665	
  
9666	
  
9667	
  
9668	
  
9669	
  
9670	
  
9671	
  
9672	
  
9673	
  
9674	
  
9675	
  
9676	
  
9677	
  
9678	
  
9679	
  
9680	
  
9681	
  
9682	
  
9683	
  
9684	
  
9685	
  
9686	
  
9687	
  
9688	
  
9689	
  
9690	
  
9691	
  
9692	
  
9693	
  
9694	
  
9695	
  
9696	
  
9697	
  
9698	
  
9699	
  
9700	
  
9701	
  
9702	
  
9703	
  
9704	
  
9705	
  

Carter, R.A., Wicks, I.P., 2001. Vascular cell adhesion molecule 1(CD-106): a multifaceted regulatorof joint inflammation. Arthritis Rheum. 44, 985-994.
Casarett, G.W., 1973. Pathogenesis of radionuclide-induced tumors, Radionuclide Carcinogenesis.
National Technical Information Service, Springfield, pp. 1-14.
Casas, S., Ollila, J., Aventin, A., et al., 2003. Changes in apoptosis-related pathways in acute
myelocytic leukemia. Cancer Genet. Cytogenet. 146, 89-101.
Castiglioni, F., Terenziani, M., Carcangiu, M.L., et al., 2007. Radiation effects on development of
HER2-positive breast carcinomas. Clin. Cancer. Res. 13, 46-51.
Catlin, S.N., Busque, L., Gale, R.E., et al., 2011. The replication rate of human hematopoietic stem
cells in vivo. Blood 117, 4460-4466.
Caughey, R.W., Michels, K.B., 2009. Birth weight and childhood leukemia: a meta-analysis andreview of the current evidence. Int. J. Cancer 124, 2658-2670.
Cavalieri, E., Rogan, E., 2006. Catechol quinones of estrogens in the initiation of breast, prostate, andother human cancers: keynote lecture. Ann. N.Y. Acad. Sci. U.S.A. 1089, 286-301.
Cengiz, M., Celebioglu, B., Ozyar, E., et al., 2004. Unusual hypersensitivity to radiation therapy in apatient with dyskeratosis congenita syndrome. Oral Oncol. 40, 758-759.
Cerfolio, R.J., Bryant, A.S., Scott, E., et al., 2006. Women with pathologic stage I, II, and III nonsmall cell lung cancer have better survival than men. Chest 130, 1796-1802.
Chadwick, K.H., Leenhouts, H.P., Laheij, G.M.H., et al., 1995. The implications of a two-mutation
carcinogenesis model for internal emitters. In: van Kaick, G., Karaoglou, A., Kellerer, A.M.
(Eds.), Health Effects of Internally Deposited Radionuclides: Emphasis on Radium andThorium. World Scientific, Singapore, pp. 353-360.
Chamberlain, G., Fox, J., Ashton, B., et al., 2007. Concise review: mesenchymal stem cells: theirphenotype, differentiation capacity, immunological features, and potential for homing. StemCells 25, 2739-2749.
Chambers, S.M., Shaw, C.A., Gatza, C., et al., 2007. Aging hematopoietic stem cells decline infunction and exhibit epigenetic dysregulation. PLoS Biology 5, e201.
Charles, M.W., 2007. Radon exposure of the skin: I. Biological effects. J. Radiol. Prot. 27, 231-252.
Charruyer, A., Barland, C.O., Yue, L., et al., 2009. Transit-amplifying cell frequency and cell cycle
kinetics are altered in aged epidermis. J. Invest. Dermatol. 129, 2574-2583.
Chen K.Y., Withers H.R., 1972. Survival characteristics of stem cells of gastric mucosa in C3H micesubjected to localized gamma irradiation. Int. J. Radiat Biol. 21, 521-534.
Chen, M.F., Lin, C.T., Chen, W.C., et al., 2006. The sensitivity of human mesenchymal stem cells toionizing radiation. Int. J. Rad. Onc. Biol. Phys. 66, 244-253.
Chen, M.S., Woodward, W.A., Behbod, F., et al., 2007. Wnt/beta-catenin mediates radiation
resistance of Sca1+ progenitors in an immortalized mammary gland cell line. J. Cell. Sci. 120,468-477.
Cheng, J., Turkel, N., Hemati, N., et al., 2008. Centrosome misorientation reduces stem cell division
during ageing. Nature 456, 599-604.
Cheng, T., Rodrigues, N., Shen, H., et al., 2000. Hematopoietic stem cell quiescence maintained by
p21cip1/waf1. Science 287, 1804-1808.
Chepko, G., Dickson, R.B., 2003. Ultrastructure of the putative stem cell niche in rat mammary
epithelium. Tissue & Cell 35, 83-93.
Chepko, G., Smith, G.H., 1997. Three division-competent, structurally-distinct cell populations
contribute to murine mammary epithelial renewal. Tissue Cell 29, 239-253.
Chiosea, S., Jelezcova, E., Chandran, U., et al., 2007. Overexpression of Dicer in precursor lesions oflung adenocarcinoma. Cancer Res. 67, 2345-2350.
Chokkalingam, A.P., Metayer, C., Scelo, G., et al., 2012. Fetal growth and body size genes and risk ofchildhood acute lymphoblastic leukemia. Cancer Causes & Control: 23, 1577-1585.
Chopra, D.P., Flaxman, B.A., 1974. Comparative proliferative kinetics of cells from normal humanepidermis and benign epidermal hyperplasia (psoriasis) in vitro. Cell and Tissue Kinetics 7, 6976.

232

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9706	
  
9707	
  
9708	
  
9709	
  
9710	
  
9711	
  
9712	
  
9713	
  
9714	
  
9715	
  
9716	
  
9717	
  
9718	
  
9719	
  
9720	
  
9721	
  
9722	
  
9723	
  
9724	
  
9725	
  
9726	
  
9727	
  
9728	
  
9729	
  
9730	
  
9731	
  
9732	
  
9733	
  
9734	
  
9735	
  
9736	
  
9737	
  
9738	
  
9739	
  
9740	
  
9741	
  
9742	
  
9743	
  
9744	
  
9745	
  
9746	
  
9747	
  
9748	
  
9749	
  
9750	
  
9751	
  
9752	
  
9753	
  
9754	
  
9755	
  
9756	
  
9757	
  

Christensen, R., Alsner, J., Brandt Sorensen, F., et al., 2008. Transformation of human mesenchymal
stem cells in radiation carcinogenesis: long-term effect of ionizing radiation. Regen. Med. 3,
849-861.
Christov, K., Swanson, S.M., Guzman, R.C., et al., 1993. Kinetics of mammary epithelial cellproliferation in pituitary isografted BALB/c mice. Carcinogenesis 14, 2019-2025.
Chu-Tse, W., Lajtha, L.G., 1975. Hemopoietic stem-cell kinetics during continuous irradiation. Int. J.
Radiat. Biol. Relat. Stud. Phys. Chem. Med. 27, 41-50.
Ciampi, R., Nikiforov, Y.E., 2007. RET/PTC rearrangements and BRAF mutations in thyroid
tumorigenesis. Endocrinology 148, 936-941.
Cicalese, A., Bonizzi, G., Pasi, C.E., et al., 2009. The tumor suppressor p53 regulates polarity of selfrenewing divisions in mammary stem cells. Cell 138, 1083-1095.
Clarke, R.B., Howell, A., Anderson, E., 1997a. Estrogen sensitivity of normal human breast tissue invivo and implanted into athymic nude mice: analysis of the relationship between estrogeninduced proliferation and progesterone receptor expression. Breast Cancer Res. Treat 45, 121133.
Clarke, R.B., Howell, A., Potten, C.S., et al., 1997b. Dissociation between steroid receptor expression
and cell proliferation in the human breast. Cancer Res. 57, 4987-4991.
Clarke, R.B., Spence, K., Anderson, E., et al., 2005. A putative human breast stem cell population isenriched for steroid receptor-positive cells. Develop Biol. 277, 443-456.
Claudinot, S., Nicolas, M., Oshima, H., et al., 2005. Long-term renewal of hair follicles fromclonogenic multipotent stem cells. Proc. Natl. Acad. Sci. U.S.A. 102, 14677-14682.
Clayton, E., Doupe, D.P., Klein, A.M., et al., 2007. A single type of progenitor cell maintains normal
epidermis. Nature 446, 185-189.
Clayton, H., Titley, I., Vivanco, M.D., 2004. Growth and differentiation of progenitor/stem cells
derived from the human mammary gland. Exp. Cell Res. 297, 444-460.
Cleary, H., Boulton, E., Plumb, M., 2001. Allelic loss on chromosome 4 (Lyr2/TLSR5) is associatedwith myeloid, B–lympho-myeloid, and lymphoid (B and T) mouse radiation-induced leukemias.
Blood 98, 1549-1554.
Clifton, K.H., DeMott, R.K., Mulcahy, R.T., et al., 1978. Thyroid gland formation from inocula ofmonodispersed cells: early results on quantitation, function, neoplasia and radiation effects. Int.
J. Radiat. Oncol. Biol. Phys. 4, 987-990.
Clifton, K.H., Sridharan, B.N., Douple, E.B., 1975. Mammary carcinogenesis-enhancing effect ofadrenalectomy in irradiated rats with pituitary tumor MtT-F4. J. Natl. Cancer. Inst. 55, 485-487.
Clifton, K.H., Tanner, M.A., Gould, M.N., 1986. Assessment of radiogenic cancer initiation
frequency per clonogenic rat mammary cell in vivo. Cancer Res. 46, 2390-2395.
Cobaleda, C., Jochum, W., Busslinger, M., 2007. Conversion of mature B cells into T cells bydedifferentiation to uncommitted progenitors. Nature 449, 473-477.
Coclet, J., Foureau, F., Ketelbant, P., et al., 1989. Cell population kinetics in dog and human adultthyroid. Clin. Endocrinol. 31, 655-665.
Coggle, J.E., Williams, J.P., 1990. Experimental studies of radiation carcinogenesis in the skin: areview. Int. J. Radiat. Biol. 57, 797-808.
Cooper, M., Pinkus, H., 1977. Intrauterine transplantation of rat basal cell carcinoma as a model forreconversion of malignant to benign growth. Cancer Res. 37, 2544-2552.
Cote, M.L., Kardia, S.L., Wenzlaff, A.S., et al., 2005. Risk of lung cancer among white and black
relatives of individuals with early-onset lung cancer. JAMA 293, 3036-3042.
Cotsarelis, G., Sun, T.T., Lavker, R.M., 1990. Label-retaining cells reside in the bulge area ofpilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.
Cell 61, 1329-1337.
Counter, C.M., Avilion, A.A., LeFeuvre, C.E., et al., 1992. Telomere shortening associated withchromosome instability is arrested in immortal cells which express telomerase activity. Embo J.
11, 1921-1929.
Cowland, J.B., Hother, C., Gronbaek, K., 2007. MicroRNAs and cancer. APMIS 115, 1090-1106.

233

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9758	
  
9759	
  
9760	
  
9761	
  
9762	
  
9763	
  
9764	
  
9765	
  
9766	
  
9767	
  
9768	
  
9769	
  
9770	
  
9771	
  
9772	
  
9773	
  
9774	
  
9775	
  
9776	
  
9777	
  
9778	
  
9779	
  
9780	
  
9781	
  
9782	
  
9783	
  
9784	
  
9785	
  
9786	
  
9787	
  
9788	
  
9789	
  
9790	
  
9791	
  
9792	
  
9793	
  
9794	
  
9795	
  
9796	
  
9797	
  
9798	
  
9799	
  
9800	
  
9801	
  
9802	
  
9803	
  
9804	
  
9805	
  
9806	
  
9807	
  
9808	
  
9809	
  
9810	
  

Crasta, K., Ganem, N.J., Dagher, R., et al., 2012. DNA breaks and chromosome pulverization fromerrors in mitosis. Nature 482, 53-58.
Crosnier, C., Stamataki, D., Lewis, J., 2006. Organizing cell renewal in the intestine: stem cells,
signals and combinatorial control. Nat. Rev. Genet. 7, 349-359.
Curcio, F., Ambesi-Impiombato, F.S., Perrella, G., et al., 1994. Long-term culture and functional
characterization of follicular cells from adult normal human thyroids. Proc. Nat. Acad. Sci. U S
A 91, 9004-9008.
d'Adda di Fagagna, F., Reaper, P.M., Clay-Farrace, L., et al., 2003. A DNA damage checkpoint
response in telomere-initiated senescence. Nature 426, 194-198.
da Silva Meirelles, L., Chagastelles, P.C., Nardi, N.B., 2006. Mesenchymal stem cells reside invirtually all postnatal organs and tissues. J. Cell Sci. 119, 2204-2213.
Damante, G., Tell, G., Di Lauro, R., 2001. A unique combination of transcription factors controls
differentiation of thyroid cells. Prog. Nuc. Acid Res. and Mol. Biol. 66, 307-356.
Danet, G.H., Pan, Y., Luonog, J.L., et al., 2003. Expansion of human SCID-repopulating cells underhypoxic conditions. J. Clin. Invest. 112, 126-135.
Dani, N., Olivero, M., Mareschi, K., et al., 2012. The MET oncogene transforms human primarybone-derived cells into osteosarcomas by targeting committed osteo-progenitors. J. Bone Miner.
Res. 27, 1322-1334.
Danial, N.N., 2007. BCL-2 family proteins: critical checkpoints of apoptotic cell death. Clin. Cancer
Res. 13, 7254-7263.
Daniel, C.W., Berger, J.J., Strickland, P., et al., 1984. Similar growth pattern of mouse mammary
epithelium cultivated in collagen matrix in vivo and in vitro. Dev. Biol. 104, 57-64.
Daniel, C.W., De Ome, K.B., Young, J.T., et al., 1968. The in vivo life span of normal andpreneoplastic mouse mammary glands: a serial transplantation study. Proc. Natl. Acad. Sci. U S
A 61, 53-60.
Daniel, C.W., Deome, K.B., 1965. Growth of mouse mammary glands in vivo after monolayer culture.
Science 149, 634-636.
Daniel, C.W., Silberstein, G.B., Strickland, P., 1987. Direct action of 17 beta-estradiol on mouse
mammary ducts analyzed by sustained release implants and steroid autoradiography. Cancer
Res. 47, 6052-6057.
Daniel, C.W., Young, L.J., Medina, D., et al., 1971. The influence of mammogenic hormones onserially transplanted mouse mammary gland. Exp. Gerontol. 6, 95-101.
Daniely, Y., Liao, G., Dixon, D., et al., 2004. Critical role of p63 in the development of a normal
esophageal and tracheobronchial epithelium. Am. J. Physiol. Cell Physiol. 287, C171-181.
Darby, S.C., Doll, R., Gill, S.K., et al., 1987. Long term mortality after a single treatment course with
X-rays in patients treated for ankylosing spondylitis. Br. J. Cancer 55, 179-190.
Darby, S.C., Hill, D., Auvinen, A., et al., 2005. Radon in homes and risk of lung cancer: collaborative
analysis of individual data from 13 European case-control studies. Br. Med. J. (Clin Res
Edition) 330, 223.
Darby S., Hill D., Deo H., et al., 2006. Residential radon and lung cancer--detailed results of acollaborative analysis of individual data on 7148 persons with lung cancer and 14,208 persons
without lung cancer from 13 epidemiologic studies in Europe. Scand. J. Work Environ. Health.
32 Suppl 1, 1-83.
Darby, S.C., Reeves, G., Key, T., et al., 1994. Mortality in a cohort of women given X-ray therapy for
metropathia haemorrhagica. Int. J. Cancer 56, 793-801.
Davis, F.G., Boice, J.D., Jr., Hrubec, Z., et al., 1989. Cancer mortality in a radiation-exposed cohortof Massachusetts tuberculosis patients. Cancer Res. 49, 6130-6136.
Davis, L.A., Zur Neiden, N.I., 2008. Mesodermal fate decision of a stem cell: the Wnt switch. Cell
Mol. Live Sci. 65, 2658-2674.
De Felice, M., Di Lauro, R., 2004. Thyroid development and its disorders: genetics and molecularmechanisms. Endocr. Rev. 25, 722-746.
De Felice, M., Ovitt, C., Biffali, E., et al., 1998. A mouse model for hereditary thyroid dysgenesis andcleft palate. Nat. Genet. 19, 395-398.

234

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9811	
  
9812	
  
9813	
  
9814	
  
9815	
  
9816	
  
9817	
  
9818	
  
9819	
  
9820	
  
9821	
  
9822	
  
9823	
  
9824	
  
9825	
  
9826	
  
9827	
  
9828	
  
9829	
  
9830	
  
9831	
  
9832	
  
9833	
  
9834	
  
9835	
  
9836	
  
9837	
  
9838	
  
9839	
  
9840	
  
9841	
  
9842	
  
9843	
  
9844	
  
9845	
  
9846	
  
9847	
  
9848	
  
9849	
  
9850	
  
9851	
  
9852	
  
9853	
  
9854	
  
9855	
  
9856	
  
9857	
  
9858	
  
9859	
  
9860	
  
9861	
  
9862	
  
9863	
  

De Felice, M., Postiglione, M.P., Di Lauro, R., 2004. Minireview: thyrotropin receptor signaling indevelopment and differentiation of the thyroid gland: insights from mouse models and humandiseases. Endocrinol. 145, 4062-4067.
de Visser, K.E., Ciampricotti, M., Michalak, E.M., et al., 2012. Developmental stage-specificcontribution of Lgr5(+) cells to basal and luminal epithelial lineages in the postnatal mammary
gland. J. Pathol. 228, 300-309.
Decosse, J.J., Gossens, C.L., Kuzma, J.F., et al., 1973. Breast cancer: Induction of differentiation byembryonic tissue. Science 181, 1057-1058.
Deininger, M.W., Bose, S., Gora-Tybor, J., et al., 1998. Selective induction of leukemia-associatedfusion genes by high-dose ionizing radiation. Cancer Res. 58, 421-425.
Demidchik, Y.E., Saenko, V.A., Yamashita, S., 2007. Childhood thyroid cancer in Belarus, Russia,
and Ukraine after Chernobyl and at present. Arq. Bras. Endocrinol. Metab. 51, 748-762.
Denissenko, M.F., Chen, J.X., Tang, M.S., et al., 1997. Cytosine methylation determines hot spots of
DNA damage in the human P53 gene. Proc. Natl. Acad. Sci. U.S.A. 94, 3893-3898.
Dennis, J.E., Charbord, P., 2002. Origin and differentiation of human and murine stroma. Stem Cells
20, 205-214.
DeOme, K.B., Faulkin, L.J., Jr., Bern, H.A., et al., 1959. Development of mammary tumors fromhyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H
mice. Cancer Res. 19, 515-520.
DeOme, K.B., Miyamoto, M.J., Osborn, R.C., et al., 1978. Detection of inapparent nodule
transformed cells in the mammary gland tissues of virgin female BALB/cfC3H mice. Cancer
Res. 38, 2103-2111.
DeRose, C., Claycamp, H.G., 1989. Dimethylformamide-induced changes in the radiation survival oflow- and high-passage intestinal epithelial cells (IEC-17) in vitro. Radiat Res. 118, 269-282.
Desai, P., Perino, G., Present, D., et al., 1996. Sarcoma in association with bone infarcts. Report offive cases. Pathol. & Lab. Med. 120, 482-489.
Deugnier, M.A., Faraldo, M.M., Janji, B., et al., 2002. EGF controls the in vivo developmental
potential of a mammary epithelial cell line possessing progenitor properties. J. Cell Biol. 159,
453-463.
Devi, P.U., Satyamitra, M., 2005. Tracing radiation induced genomic instability in vivo in the
haemopoietic cells from fetus to adult mouse. Br. J. Radiol. 78, 928-933.
Di Como, C.J., Urist, M.J., Babayan, I., et al., 2002. p63 expression profiles in human normal andtumor tissues. Clin. Cancer Res. 8, 494-501.
Di Majo, V., Rebessi, S., Pazzaglia, S., et al., 2003. Carcinogenesis in laboratory mice after low dosesof ionizing radiation. Radiat. Res. 159, 102-108.
Diallo, R., Schaefer, K.L., Poremba, C., et al., 2001. Monoclonality in normal epithelium and in
hyperplastic and neoplastic lesions of the breast. J. Pathol. 193, 27-32.
Dibble, R., Langeburg, W., Bair, S., et al., 2005. Natural history of non-small cell lung cancer in nonsmokers, J. Clin. Oncol., p. 7252.
Dick, J.E., 2003. Breast cancer stem cells revealed. Proc. Natl. Acad. Sci. U S A. 100, 3547-3549.
Ding, L., Getz, G., Wheeler, D.A., et al., 2008. Somatic mutations affect key pathways in lungadenocarcinoma. Nature 455, 1069-1075.
Doll, R., Wakeford, R., 1997. Risk of childhood cancer from fetal irradiation. Br. J. Radiol. 70, 130139.
Dominici, M., Le Blanc, K., Mueller, I., et al., 2006. Minimal criteria for defining multipotent
mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
Cytotherapy 8, 15-17.
Dong, Y., Mao, J.P., Liu, B., et al., 2001. [Variant frequency of erythrocyte glycophorin A in persons
accidentally irradiated by (60)Co source]. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 9, 174-177.
Dontu, G., Abdallah, W.M., Foley, J.M., et al., 2003. In vitro propagation and transcriptional profilingof human mammary stem/progenitor cells. Genes & Development 17, 1253-1270.
Dontu, G., Jackson, K., McNicholas, E., et al., 2004. Role of Notch signaling in cell-fate
determination of human mammary stem/progenitor cells. Breast Cancer Res. 6, R605 - R615.

235

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9864	
  
9865	
  
9866	
  
9867	
  
9868	
  
9869	
  
9870	
  
9871	
  
9872	
  
9873	
  
9874	
  
9875	
  
9876	
  
9877	
  
9878	
  
9879	
  
9880	
  
9881	
  
9882	
  
9883	
  
9884	
  
9885	
  
9886	
  
9887	
  
9888	
  
9889	
  
9890	
  
9891	
  
9892	
  
9893	
  
9894	
  
9895	
  
9896	
  
9897	
  
9898	
  
9899	
  
9900	
  
9901	
  
9902	
  
9903	
  
9904	
  
9905	
  
9906	
  
9907	
  
9908	
  
9909	
  
9910	
  
9911	
  
9912	
  
9913	
  
9914	
  
9915	
  

Dorfman, H.D., Czerniak, B., 1998. Bone Tumors. Mosby, St. Louis, USA.
Dove, W.F., Cormier, R.T., Gould, K.A., et al., 1998. The intestinal epithelium and its neoplasms:
genetic, cellular and tissue interactions. Phil. Trans. Roy. Soc. 353, 915-923.
Dover, R., Potten, C.S., 1983. Radiosensitivity of normal human epidermal cells in culture. Int. J.
Radiat. Biol. & Rel. Stud. Phys. Chem. Med. 43, 681-685.
Du, Y.X., Cha, Q., Chen, X.W., et al., 1996. An epidemiological study of risk factors for lung cancer
in Guangzhou, China. Lung Cancer 14 Suppl 1, S9-37.
Ducy, P., Schinke, T., Karsenty, G., 2000. The osteoblast: a sophisticated fibroblast under central
surveillance. Science 289, 1501-1504.
Dutreix, J., 1986. Human skin: early and late reactions in relation to dose and its time distribution. Br.
J. Radiol. Suppl. 19, 22-28.
Dutu, T., Michiels, S., Fouret, P., et al., 2005. Differential expression of biomarkers in lungadenocarcinoma: a comparative study between smokers and never-smokers. Ann. Oncol. 16,
1906-1914.
Eberhard, D.A., Johnson, B.E., Amler, L.C., et al., 2005. Mutations in the epidermal growth factor
receptor and in Kras are predictive and prognostic indicators in patients with non-small-celllung cancer treated with chemotherapy alone and in combination with erlotinib. J. Clin. Oncol.
23, 5900-5909.
Eckerman, K.F., 1995. Internal dosimetry schemes, their physical, biological and epidemiologicalbasis. In: van Kaick, G., Karaoglou, A., Kellerer, A.M. (Eds.), Health Effects of InternallyDeposited Radionuclides: Emphasis on Radium and Thorium. World Scientific, Singapore, pp.
9-18.
Eckfeldt, C.E., Mendenhall, E.M., Flynn, C.M., et al., 2005. Functional analysis of humanhematopoietic stem cell gene expression using zebrafish. PLoS Biol. 3, e254.
Eder, M., Scherr, M., 2005. MicroRNA and lung cancer. N. Engl. J. Med. 352, 2446-2448.
Ellender, G., Feik, S.A., Carach, B.J., 1988. Periosteal structure and development in a rat caudal
vertebra. J. Anat. 158, 173-187.
Ellender, M., Harrison, J.D., Kozlowski, R., et al., 2006. In utero and neonatal sensitivity of
ApcMin/+ mice to radiation-induced intestinal neoplasia. Int. J. Radiat. Biol. 82, 141-151.
Ellender, M., Harrison, J.D., Meijne, E., et al., 2011. Intestinal tumours induced in Apc(Min/+) miceby X-rays and neutrons. Int. J. Radiat. Biol. 87, 385-399.
Ellender, M., Harrison, J.D., Pottinger, H., et al., 2001. Induction of osteosarcoma and acute myeloid
leukaemia in CBA/H mice by the alpha-emitting nuclides, uranium-233, plutonium-239 andamericium-241. Int. J. Radiat. Biol. 77, 41-52.
EPA, 2011. EPA radiogenic cancer risk models and projections for the U.S. population. U.S.
Environmental Protection Agency publication 402-R-11-001. Washington, DC, USA.
Epperly, M.W., Bray, J.A., Carlos, T.M., et al., 1999. Biology of marrow stromal cell lines derived
from long-term bone marrow cultures of Trp53-deficient mice. Radiat. Res. 152, 29-40.
Epstein, E.H., 2008. Basal cell carcinomas: attack of the hedgehog. Nat. Rev. Cancer 8, 743-754.
Epstein, W.L., Maibach, H.I., 1965. Cell renewal in human epidermis. Arch. Dermatol. 92, 462-468.
Eriksen, E.F., 2010. Cellular mechanisms of bone remodeling. Rev. Endocr. Metab. Disord. 11, 219227.
Essers, J., van Steeg, H., de Wit, J., et al., 2000. Homologous and non-homologous recombinationdifferentially affect DNA damage repair in mice. Embo. J. 19, 1703-1710.
Esteller, M., 2007. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat. Rev.
Genet. 8, 286-298.
Ethier, S.P., Adams, L.M., Ullrich, R.L., 1984. Morphological and histological characteristics ofmammary dysplasias occurring in cell dissociation-derived murine mammary outgrowths.
Cancer Res. 44, 4517-4522.
Ethier, S.P., Cundiff, K.C., 1987. Importance of extended growth potential and growth factor
independence on in vivo neoplastic potential of primary rat mammary carcinoma cells. Cancer
Res. 47, 5316-5322.

236

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9916	
  
9917	
  
9918	
  
9919	
  
9920	
  
9921	
  
9922	
  
9923	
  
9924	
  
9925	
  
9926	
  
9927	
  
9928	
  
9929	
  
9930	
  
9931	
  
9932	
  
9933	
  
9934	
  
9935	
  
9936	
  
9937	
  
9938	
  
9939	
  
9940	
  
9941	
  
9942	
  
9943	
  
9944	
  
9945	
  
9946	
  
9947	
  
9948	
  
9949	
  
9950	
  
9951	
  
9952	
  
9953	
  
9954	
  
9955	
  
9956	
  
9957	
  
9958	
  
9959	
  
9960	
  
9961	
  
9962	
  
9963	
  
9964	
  
9965	
  
9966	
  
9967	
  
9968	
  

Ethier, S.P., Ullrich, R.L., 1982. Detection of ductal dysplasia in mammary outgrowths derived fromcarcinogen-treated virgin female BALB/c mice. Cancer Res. 42, 1753-1760.
Evans, M.J., Kaufman, M.H., 1981. Establishment in culture of pluripotential cells from mouseembryos. Nature 292, 154-156.
Evans, M.J., Shami, S.G., Cabral-Anderson, L.J., et al., 1986. Role of nonciliated cells in renewal of
the bronchial epithelium of rats exposed to NO2. Am. J. Pathol. 123, 126-133.
Evans, M.J., Van Winkle, L.S., Fanucchi, M.V., et al., 2001. Cellular and molecular characteristics ofbasal cells in airway epithelium. Exp. Lung Res. 27, 401-415.
Ewan, K.B., Shyamala, G., Ravani, S.A., et al., 2002. Latent TGF-beta activation in mammary gland:
Regulation by ovarian hormones affects ductal and alveolar proliferation. Am. J. Path. 160,
2081-2093.
Ewan, K.B.R., Oketch-Rabah, H.A., Ravani, S.A., et al., 2005. Proliferation of estrogen receptoralpha-positive mammary epithelial cells is restrained by transforming growth factor-beta 1 inadult mice. Am. J. Pathol. 167, 409-417.
Ezashi, T., Das, P., Roberts, R.M., 2005. Low O2 tensions and the prevention of differentiation of hES
cells. Proc. Natl. Acad. Sci. U S A. 102, 4783-4788.
Fagin, J.A., 2004. Challenging dogma in thyroid cancer molecular genetics-role of RET/PTC and
BRAF in tumor initiation. J. Clin. Endocrinol. Metab. 89, 4264-4266.
Fagin, J.A., Mitsiades, N., 2008. Molecular pathology of thyroid cancer: diagnostic and clinicalimplication. Best. Pract. Res. Clin. Endocrinol. Metab. 22, 955-968.
Falconer, E., Chavez, E.A., Henderson, A., et al., 2010. Identification of sister chromatids by DNA
template strand sequences. Nature 463, 93-97.
Fan, H., Oro, A.E., Scott, M.P., et al., 1997. Induction of basal cell carcinoma features in transgenic
human skin expressing Sonic Hedgehog. Nat. Med 3, 788-792.
Farber, E., 1984. Pre-cancerous steps in carcinogenesis. Their physiological adaptive nature. Biochem.
Biophys. Acta 738, 171-180.
Farber, I., 1987. Possible etiologic mechanisms in chemical carcinogenesis. Env. Health Persp. 75,
65-70.
Fawcett, D.W., 1986. A Textbook of Histology. W.B. Saunders Co., Philadelphia, USA.
Fazaa, B., Cribier, B., Zaraa, I., et al., 2007. Low-dose X-ray depilatory treatment induces
trichoblastic tumors of the scalp. Dermatology 215, 301-307.
Feinendegen, L.E., 1985. Microdosimetric approach to raw analysis of cell responses at low dose andlow dose rate. Radiat. Prot. Dosimetry 13, 299-306.
Fenton, C.L., Lukes, Y., Nicholson, D., et al., 2000. The ret/PTC mutations are common in sporadic
papillary thyroid carcinoma of children and young adults. J. Clin. Endocrinol. Metab. 85, 11701175.
Ferlay, J., Shin, H.R., Bray, F., et al., 2010. GLOBOCAN 2008, Cancer Incidence and Mortality
Worldwide. IARC Cancer Base No. 10 [Internet]. 2010 edition. International Agency for
Research on Cancer, Lyon, France.
Fernandez-Gonzalez, R., Illa-Bochaca, I., Shelton, D.N., et al., 2010. In situ analysis of cell
populations: long-term label-retaining cells. Methods Mol. Biol. 621.
Fernandez-Gonzalez, R., Illa-Bochaca, I., Welm, B.E., et al., 2009. Mapping mammary glandarchitecture using multi-scale in situ analysis. Integr. Biol. 1, 80 - 89.
Fibbe, W.E., Noort, W.A., 2003. Mesenchymal stem cells and hematopoietic stem cell transplantation.
Ann. New York Acad. Sci. 996, 235-244.
Field, J.B., MacCammon, C.J., Valentine, R.J., et al., 1959. Failure of radioiodine to induce thyroid
cancer in the rat. Cancer Res 19, 870-873.
Fierabracci, A., Puglisi, M.A., Giuliani, L., et al., 2008. Identification of an adult stem/progenital celllike population in the human thyroid. J. Endocrinol. 198, 471-487.
Finak, G., Bertos, N., Pepin, F., et al., 2008. Stromal gene expression predicts clinical outcome inbreast cancer. Nat. Med. 14, 518-527.
Fisher, D.A., Dussault, J., 1974. Development of the mammalian thyroid gland. Handbook on
physiology. Am. Physiol. Society, Washington, D.C., pp. 21-38.

237

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
9969	
  
9970	
  
9971	
  
9972	
  
9973	
  
9974	
  
9975	
  
9976	
  
9977	
  
9978	
  
9979	
  
9980	
  
9981	
  
9982	
  
9983	
  
9984	
  
9985	
  
9986	
  
9987	
  
9988	
  
9989	
  
9990	
  
9991	
  
9992	
  
9993	
  
9994	
  
9995	
  
9996	
  
9997	
  
9998	
  
9999	
  
10000	
  
10001	
  
10002	
  
10003	
  
10004	
  10005	
  
10006	
  
10007	
  
10008	
  
10009	
  10010	
  10011	
  10012	
  10013	
  10014	
  10015	
  
10016	
  10017	
  10018	
  10019	
  10020	
  Fitzgerald, T.J., Henault, S., Santucci, M.A., et al., 1989. Recombinant murine GM-CSF increase
resistance of some factor dependent hematopoietic progenitor cells in low-dose rate gamma
irradiation. Int. J. Radiat. Oncol. Biol. Phys. 17, 323-335.
Fliedner, T.M., 1998. The role of blood stem cells in hematopoietic cell renewal. Stem Cells 16, 13-29.
Fliedner, T.M., Graessle, D., Paulsen, C., et al., 2002. Structure and function of bone marrow
hemopoiesis: mechanisms of response to ionizing radiation exposure. Cancer Biother.
Radiopharm. 17, 405-426.
Flores, I., Benetti, R., Blasco, M.A., 2006. Telomerase regulation and stem cell behaviour. Current
Opinion in Cell Biol. 18, 254-260.
Flores, I., Blasco, M.A., 2009. A p53-dependent response limits epidermal stem cell functionality andorganismal size in mice with short telomeres. PloS one 4, e4934.
Flores, I., Canela, A., Vera, E., et al., 2008. The longest telomeres: a general signature of adult stem
cell compartments. Genes & Development 22, 654-667.
Fodde, R., Smits, R., Clevers, H., 2001. APC, signal transduction and genetic instability in colorectal
cancer. Nat. Rev. Cancer 1, 55-67.
Fortunel, N.O., Cadio, E., Vaigot, P., et al., 2010. Exploration of the functional hierarchy of the basal
layer of human epidermis at the single-cell level using parallel clonal microcultures ofkeratinocytes. Exp. Dermatol. 19, 387-392.
Fortunel, N.O., Hatzfeld, J.A., Rosemary, P.A., et al., 2003a. Long-term expansion of human
functional epidermal precursor cells: promotion of extensive amplification by low TGF-beta1
concentrations. J. Cell. Sci. 116, 4043-4052.
Fortunel, N.O., Otu, H.H., Ng, H.H., et al., 2003b. Comment on " 'Stemness': transcriptional profilingof embryonic and adult stem cells" and "a stem cell molecular signature." Science 302, 393;
author reply 393.
Francois, S., Bensidhoum, M., Mouiseddine, M., et al., 2006. Local irradiation not only induceshoming of human mesenchymal stem cells at exposed sites but promotes their widespreadengraftment to multiple organs: a study of their quantitative distribution after irradiation
damage. Stem Cells 24, 1020-1029.
Freedman, V.H., Shin, S.I., 1974. Cellular tumorigenicity in nude mice: correlation with cell growthin semi-solid medium. Cell 3, 355-359.
Friedberg, E.C., Walker, G.C., Siede, W., 2006. DNA repair and mutagenesis. American Soc. forMicrobiology Press, Washington, D.C.
Frieben, A., 1902. Desmonstration eines cabcroids des rechten handriickens das dich nach
langdauernder einwirking von Rotgenstrahlen entwickelt hatte. Fortschr. Get. Roentgenol. 7,
106.
Friedenstein, A.J., Chailakhjan, R.K., Lalykina, K.S., 1970. The development of fibroblast colonies inmonolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet. 3, 393-403.
Friedenstein, A.J., Chailakhyan, R.K., Gerasimov, U.V., 1987. Bone marrow osteogenic stem cells: invitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet. 20, 263-272.
Friedenstein, A.J., Latzinik, N.V., Gorskaya, U.F., et al., 1981. Radiosensitivity and postirradiation
changes of bone marrow clonogenic stromal mechanocytes. Int. J. Radiat. Biol. Relat. Stud.
Phys. Chem. Med. 39, 537-546.
Friend, S.H., Bernards, R., Rogelj, S., et al., 1986. A human DNA segment with properties of the genethat predisposes to retinoblastoma and osteosarcoma. Nature, 643-646.
Frost, H.M., 1963. Bone remodeling dynamics. In: Lam, C.R. (Ed.). Charles C. Thomas, Springfield,
Illinois, USA.
Frost, H.M., 1980. Skeletal physiology and bone remodeling. J.B. Lippincott Co., Philadelphia.
Fry, R.J.M., Powers-Risius, P., Alpen, E.L., et al., 1983. High-LET radiation carcinogenesis. Adv.
Space Res. 3, 241-248.
Fry, S.A., 1998. Studies of U.S. radium dial workers: an epidemiological classic. Radiat. Res. 150,
S21-29.
Fuchs, E., 2008. Skin stem cells: rising to the surface. J. Cell Biol. 180, 273-284.

238

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10021	
  
10022	
  
10023	
  
10024	
  
10025	
  
10026	
  10027	
  
10028	
  
10029	
  10030	
  10031	
  
10032	
  10033	
  10034	
  10035	
  
10036	
  10037	
  10038	
  10039	
  10040	
  
10041	
  
10042	
  10043	
  10044	
  10045	
  
10046	
  10047	
  10048	
  10049	
  10050	
  10051	
  10052	
  10053	
  10054	
  10055	
  10056	
  10057	
  10058	
  10059	
  10060	
  10061	
  
10062	
  
10063	
  
10064	
  
10065	
  10066	
  
10067	
  
10068	
  
10069	
  10070	
  10071	
  
10072	
  10073	
  Fuchs, E., Tumbar, T., Guasch, G., 2004. Socializing with the neighbors: stem cells and their niche.
Cell 116, 769-778.
Fugazzola, L., Pilotti, S., Pinchera, A.,, 1995. Oncogenic rearrangements of the RET proto-oncogene
in papillary thyroid carcinomas from children exposed to the Chernobyl nuclear accident.
Cancer Res. 55, 5617-5620.
Fujii, H., Cunha, G.R., Norman, J.T., 1982. The induction of adenocarinomatous differentiation in
neoplastic bladder epithelium by an embryonic prostatic inducer. J. Urol. 128, 858-861.
Fujimitsu, Y., Nakanishi, H., Inada, K., et al., 1996. Development of aberrant crypt foci involves afission mechanism as revealed by isolation of aberrant crypts. J. Cancer Res. 87, 1199-1203.
Fukuda, K., Kikuchi, Y., 2005. Endoderm development in vertebrates: fate mapping, induction andregional specification. Dev. Growth Differ. 47, 343-355.
Furukawa, K., Preston, D., Funamoto, S., et al., 2013. Long-term trend of thyroid cancer risk among
Japanese atomic-bomb survivors: 60 years after exposure. Int. J. Cancer 132, 1222-1226.
Furukawa, K., Preston, D.L., Lonn, S., et al., 2010. Radiation and smoking effects on lung cancer
incidence among atomic bomb survivors. Radiat Res. 174, 72-82.
Fusco, A., Grieco, M., Santoro, M., et al., 1987. A new oncogene in human thyroid pappilary
carcinomas and their lymph-nodal metastasis. Nature 328, 170-172.
Gallagher, R.P., Bajdik, C.D., Fincham, S., et al., 1996. Chemical exposures, medical history, and riskof squamous and basal cell carcinoma of the skin. Cancer Epidemiol. Biomarkers Prev. 5, 419424.
Galleani, J., Miranda, C., Pierotti, M.A., et al., 2009. H2AX phosphorylation and kinetics of radiationinduced DNA double strand break repaired in human primary thyrocytes. Thyroid 19, 257-264.
Gallini, R., Hendry J.H., Molineux G., et al., 1988. The effect of low dose rate on recovery ofhemopoietic and stromal progenitor cells in gamma-irradiated mouse bone marrow. Radiat. Res.
115, 481-487.
Gandhi, M., Evdokimova, V., Nikiforov, Y.E., et al., 2010. Mechanisms of chromosomal
rearrangements in solid tumors: the model of papillary thyroid carcinoma. Mol. Cell.
Endocrinol. 321, 36-43.
Garnis, C., Lockwood, W.W., Vucic, E., et al., 2006. High resolution analysis of non-small cell lungcancer cell lines by whole genome tiling path array CGH. Int. J. Cancer 118, 1556-1564.
Garte, S.J., Burns, F.J., Ashkenazi-Kimmel, T., et al., 1990. Amplification of the c-myc oncogene
during progression of radiation-induced rat skin tumors. Cancer Res. 50, 3073-3077.
Gazdar, A.F., Shigematsu, H., Herz, J., et al., 2004. Mutations and addiction to EGFR: the Achilles'heal' of lung cancers? Trends Mol. Med. 10, 481-486.
Gazzeri, S., Brambilla, E., Caron de Fromentel, C., et al., 1994. p53 genetic abnormalities and myc
activation in human lung carcinoma. Int. J. Cancer 58, 24-32.
Gazzeri, S., Brambilla, E., Chauvin, C., et al., 1990. Analysis of the activation of the myc family
oncogene and of its stability over time in xenografted human lung carcinomas. Cancer Res. 50,
1566-1570.
Gazzeri, S., Della Valle, V., Chaussade, L., et al., 1998. The human p19ARF protein encoded by thebeta transcript of the p16INK4a gene is frequently lost in small cell lung cancer. Cancer Res. 58,
3926-3931.
George, R.J., Sturmoski, M.A., May, R., et al., 2009. Loss of p21Waf1/Cip1/Sdi1 enhances intestinal
stem cell survival following radiation injury. Am. J. Physiol. Gastrointest. Liver Physiol. 296,
G245-254.
Germeshausen, M., Skokowa, J., Ballmaier, M., et al., 2008. G-CSF receptor mutations in patientswith congenital neutropenia. Curr. Opin. Hematol. 15, 332-337.
Giangreco, A., Reynolds, S.D., Stripp, B.R., 2002. Terminal bronchioles harbor a unique airway stem
cell population that localizes to the bronchoalveolar duct junction. Am. J. Pathol. 161, 173-182.
Giannakis, M., Stappenbeck, T.S., Mills, J.C., et al., 2006. Molecular properties of adult mousegastric and intestinal epithelial progenitors in their niches. J. Biol. Chem. 281, 11292-11300.
Gidali, J., 2002. Effects of protracted and chronic irradiation on the hemopoietic system in the mouse.
Br. Inst. Radiol. Supplement, London, UK.

239

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10074	
  10075	
  
10076	
  10077	
  10078	
  10079	
  10080	
  
10081	
  
10082	
  
10083	
  
10084	
  
10085	
  10086	
  10087	
  10088	  
10089	
  10090	
  
10091	
  
10092	
  
10093	
  
10094	
  
10095	
  
10096	
  
10097	
  
10098	
  10099	
  10100	
  10101	
  
10102	
  
10103	
  
10104	
  
10105	
  
10106	  
10107	  
10108	  
10109	
  10110	
  10111	
  10112	
  10113	  10114	  10115	  10116	  10117	  10118	  10119	
  10120	
  10121	  
10122	  10123	  10124	
  Gilliland, F.D., Hunt, W.C., Archer, V.E., et al., 2000. Radon progeny exposure and lung cancer risk
among non-smoking uranium miners. Health Phys. 79, 365-372.
Girard, L., Zochbauer-Muller, S., Virmani, A.K., et al., 2000. Genome-wide allelotyping of lungcancer identifies new regions of allelic loss, differences between small cell lung cancer andnon-small cell lung cancer, and loci clustering. Cancer Res. 60, 4894-4906.
Goetz, W., Morgan, W.F., Baulch, J., 2011. The effect of radiation quality on genomic DNA
methylation profiles in irradiated human cell lines. Radiat Res., 575-587.
Goff, J.P., Shields, D.S., Boggs, S.S., et al., 1997. Effects of recombinant cytokines on colony
formation by irradiated human cord blood CD34+ hematopoietic progenitor cells. Radiat Res.
147, 61-69.
Gonzalez-Suarez, E., Samper, E., Flores, J.M., et al., 2000. Telomerase-deficient mice with short
telomeres are resistant to skin tumorigenesis. Nature Genetics 26, 114-117.
Goodell, M.A., Brose, K., Paradis, G., et al., 1996. Isolation and functional properties of murine
hematopoietic stem cells that are replicating in vivo. J. Exp. Med. 183, 1797-1806.
Goodell, M.A., Rosenzweig, M., Kim, H., et al., 1997. Dye efflux studies suggest that hematopoietic
stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat.
Med. 3, 1337-1345.
Goodhead, D.T., 1985. Saturable repair models of radiation action in mammalian cells. Radiat. Res. 8,
S58-67.
Goodman, M.T., Gurney, J.G., Smith, M.A., et al., 1999. Cancer incidence and survival amongchildren and adolescents: Sympathetic nervous system tumors. In: Ries, L., Smith, M.A.,
Gurney, L.G., Linet, M., Tamra, T., Young, J.L., Bunin, G.R. (Eds.), Cancer Incidence andSurvival among Children and Adolescents: United States SEER program 1975-1995. National
Cancer Institute, Bethesda, MD.
Gorgoulis, V.G., Vassiliou, L.V., Karakaidos, P., et al., 2005. Activation of the DNA damage
checkpoint and genomic instability in human precancerous lesions. Nature 434, 907-913.
Gori, F., Hofbauer, L.C., Dunstan, C.R., et al., 2000. The expression of osteoprotegerin and RANK
ligand and the support of osteoclast formation by stromal-osteoblast lineage cells isdevelopmentally regulated. Endocrinology 141, 4768-4776.
Gossner, W., 1986. Pathology of radiation-induced bone tumors. Leukemia Res. 10, 897-904.
Gossner, W., 1999. Pathology of radium-induced bone tumors: new aspects of histopathology andhistogenesis. Radiat. Res. 152, S12-15.
Gossner, W., 2003. Target cells in internal dosimetry. Radiat. Prot. Dosim. 105, 39-42.
Gossner, W., Masse, R., Stather, J., 2000. Cells at risk for dosimetric modelling relevant to bonetumour induction. Radiat. Prot. Dosim. 92, 209-213.
Gossner, W., Wick, R.R., Spiess, H., 1995. Histopathological review of radium-224 induced bonesarcomas. In: van Kaick, G., Karaoglou, A., Kellerer, A.M. (Eds.), Health Effects of InternallyDeposited Radionuclides: Emphasis on Radium and Thorium. World Scientific, Singapore, pp.
255-259.
Gould, E., Reeves, A.J., Graziano, M.S., et al., 1999. Neurogenesis in the neocortex of adult primates.
Science 286, 548-552.
Gould, M.N., Biel, W.F., Clifton, K.H., 1977. Morphological and quantitative studies of glandformation from inocula of monodispersed rat mammary cells. Exp. Cell. Res. 107, 405-416.
Gould, M.N., Cathers, L.E., Clifton, K.H., et al., 1984. The influence of in situ repair systems on
survival of several irradiated parenchymal cell types. Br. J. Cancer Suppl 6, 191-195.
Gould, M.N., Clifton, K.H., Damiya, K., et al., 1991. Quantitative and molecular comparison of
initiation frequency of mammary carcinogenesis by radiation and chemical carcinogens. Radiat.
Env. Biophys. 30, 221-223.
Goyette, M.C., Cho, K., Fasching, C.L., et al., 1992. Progression of colorectal cancer is associatedwith multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplishedby correction of any single defect via chromosome transfer. Mol. Cell Biol. 12, 1387-1395.

240

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10125	  
10126	
  10127	  10128	  10129	
  10130	
  10131	  10132	  10133	  10134	  10135	  10136	  10137	  10138	  10139	
  10140	  10141	  10142	  10143	  10144	  10145	  10146	  10147	  10148	  10149	
  10150	
  10151	  10152	  10153	  10154	  10155	  10156	  10157	  10158	  10159	
  10160	
  10161	  
10162	  10163	  10164	  10165	
  10166	  10167	  10168	  
10169	
  10170	  10171	  10172	  10173	  10174	  10175	  10176	  10177	  Graakjaer, J., Christensen, R., Kolvraa, S., et al., 2007. Mesenchymal stem cells with high telomerase
expression do not actively restore their chromosome arm specific telomere length pattern after
exposure to ionizing radiation. BMC Mol. Biol. 8, 49.
Gragtmans, N.J., Myers, D.K., Johnson, J.R., et al., 1984. Occurrence of mammary tumors in rats
after exposure to tritium beta rays and 200-kVp X rays. Radiat Res. 99, 636-650.
Grayson, W.L., Zhao, F., Bunnell, B., et al., 2007. Hypoxia enhances proliferation and tissue
formation of human mesenchymal stem cells. Biochem Biophys. Res. Comm. 358, 948-953.
Greaves, L.C., Preston, S.L., Tadrous, P.J., et al., 2006. Mitochondrial DNA mutations are establishedin human colonic stem cells, and mutated clones expand by crypt fission. Proc. Natl. Acad. Sci.
U S A. 103, 714-719.
Green, A., Williams, G., Neale, R., et al., 1999. Daily sunscreen application and betacarotene
supplementation in prevention of basal-cell and squamous-cell carcinomas of the skin: arandomised controlled trial. Lancet 354, 723-729.
Greenberger, J.S., Eckner, R.J., Otten, J.A., et al., 1982. In vitro quantitation of lethal and physiologic
effects of total body irradiation on stromal and hematopoietic stem cells in continuous bonemarrow cultures from Rf mice. Int. J. Radiat. Oncol. Biol. Phys. 8, 1155-1165.
Greenberger, J.S., Epperly, M., 2009. Bone marrow-derived stem cells and radiation response. Semin.
Radiat. Oncol. 19, 133-139.
Gregorieff, A., Pinto, D., Begthel, H., et al., 2005. Expression pattern of Wnt signaling components inthe adult intestine. Nat. Rev. Gastroenterol. Hepatol. 129, 626-638.
Grieco, M., Santoro, M., Berlingieri, M.T., et al., 1990. PTC is a novel rearranged form of the ret
proto-oncogene and is fequently detected in vivo in human thyroid papillary carcinomas. Cell
60, 557-563.
Griem, M.L., Kleinerman, R.A., Boice, J.D., Jr., et al., 1994. Cancer following radiotherapy for peptic
ulcer. J. Natl. Cancer. Inst. 86, 842-849.
Griffin, C., Waard, H., Deans, B., et al., 2005. The involvement of key DNA repair pathways in theformation of chromosome rearrangements in embryonic stem cells. DNA Repair 4, 1019-1027.
Griffiths, D.F., Davies, S.J., Williams, D., et al., 1988. Demonstration of somatic mutation andcolonic crypt clonality by X-linked enzyme histochemistry. Nature 333, 461-463.
Griffiths, D.F., Sacco, P., Williams, D., et al., 1989. The clonal origin of experimental large bowel
tumours. Br. J. Cancer 59, 385-387.
Griffiths, S.D., Clarke, A.R., Healy, L.E., et al., 1997. Absence of p53 permits propagation of mutant
cells following genotoxic damage. Oncogene 14, 523-531.
Gronthos, S., Zannettino, A.C., Hay, S.J., et al., 2003. Molecular and cellular characterisation ofhighly purified stromal stem cells derived from human bone marrow. J. Cell. Sci. 116, 18271835.
Grosche, B., Kreuzer, M., Kreisheimer, M., et al., 2006. Lung cancer risk among German maleuranium miners: a cohort study, 1946-1998. Br. J. Cancer 95, 1280-1287.
Gross, L., 1959. [Serial cell-free passage of a radiation-activated mouse leukemia agent]. Proc. Soc.
Exp. Biol. Med. 100, 102-105.
Grosse-Gehling, P., Fargeas, C.A., Dittfeld, C., et al., 2013. CD133 as a biomarker for putative cancer
stem cells in solid tumours: limitations, problems and challenges. J. Pathol., 355-378.
Gu, Y., Jin, S., Gao, Y., et al., 1997. Ku70-deficient embryonic stem cells have increased ionizing
radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J
recombination. Proc. Natl. Acad. Sci. U S A. 94, 8076-8081.
Gudjonsson, T., Villadsen, R., Nielsen, H.L., et al., 2002. Isolation, immortalization, andcharacterization of a human breast epithelial cell line with stem cell properties. Genes Dev. 16,
693-706.
Guilmette, R.A., Muggenburg, B.A., Hahn, F.F., et al., 1987. Dosimetry of 239Pu in dogs that inhaledmonodisperse aerosols of 239PuO2. Radiat. Res. 110, 199-218.
Gundry, R.L., Boheler, K.R., Van Eyk, J.E., et al., 2008. A novel role for proteomics in the discoveryof cell-surface markers on stem cells: Scratching the surface. Proteomics. Clinical applications
2, 892-903.

241

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10178	  10179	
  10180	  
10181	  
10182	  10183	  10184	  10185	
  10186	  10187	  10188	  
10189	
  10190	  
10191	  
10192	  
10193	  
10194	  
10195	  
10196	  
10197	  
10198	
  10199	
  10200	
  10201	
  10202	
  10203	
  10204	
  10205	
  
10206	
  10207	
  10208	  10209	
  10210	
  10211	
  10212	
  10213	
  10214	
  10215	  10216	  10217	  10218	  10219	
  10220	
  10221	
  10222	
  10223	
  10224	
  10225	
  10226	
  10227	
  10228	  10229	
  10230	
  Gurley, K.E., Moser, R., Gu, Y., et al., 2009. DNA-PK suppresses a p53-independent apoptotic
response to DNA damage. EMBO reports 10, 87-93.
Hahn, F.F., McClellan, R.O., Boecker, B.B., et al., 1988. Future development of biologicalunderstanding of radiation protection: implications of nonstochastic effects. Health Phys. 55,
303-313.
Hahn, H., Wicking, C., Zaphiropoulous, P.G., et al., 1996. Mutations of the human homolog of
Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85, 841-851.
Hahn, W.C., Weinberg, R.A., 2002. Modelling the molecular circuitry of cancer. Nat. Rev. Cancer 2,
331-341.
Hamad, N.M., Elconin, J.H., Karnoub, A.E., et al., 2002. Distinct requirements for Ras oncogenesis in
human versus mouse cells. Genes Dev. 16, 2045-2057.
Hamatani, K., Eguchi, H., Ito, R., et al., 2008. RET/PTC rearrangements preferentially occurred inpapillary thyroid cancer among atomic bomb survivors exposed to high radiation dose. Cancer
Res. 68, 7176-7182.
Hammoud, Z., Tan, B., Badve, S., et al., 2008. Estrogen promotes tumor progression in a geneticallydefined mouse model of lung adenocarcinoma. Endocr. Relat. Cancer 15, 475-483.
Han, J., Kraft, P., Colditz, G.A., et al., 2006. Melanocortin 1 receptor variants and skin cancer risk. Int.
J. Cancer 119, 1976-1984.
Hanahan, D., Weinberg, R.A., 2000. The hallmarks of cancer. Cell 100, 57-70.
Hancock, S.L., Tucker, M.A., Hoppe, R.T., 1993. Breast cancer after treatment of Hodgkin's disease.
J. Natl. Cancer. Inst. 85, 25-31.
Harach, H.R., 1988. Solid cell nests of the thyroid. J. Pathol. 155, 191-200.
Harach, H.R., 1991. Thyroglobulin in human thyroid follicles with acid mucin. J. Pathol. 164, 261263.
Harada, S., Rodan, G.A., 2003. Control of osteoblast function and regulation of bone mass. Nature
423, 349-355.
Harfouche, G., Martin, M.T., 2010. Response of normal stem cells to ionizing radiation: a balance
between homeostasis and genomic stability. Mutat. Res. 704, 167-174.
Harfouche, G., Vaigot, P., Rachidi, W., et al., 2010. FGF2 Signaling is critical for DNA repair inhuman keratinocyte stem cells. Stem Cells 28, 1639-1648.
Harper, K., Lorimore, S.A., Wright, E.G., 1997. Delayed appearance of radiation-induced mutations
at the Hprt locus in murine hemopoietic cells. Exp. Hematol. 25, 263-269.
Harrington, L., 2004. Does the reservoir for self-renewal stem from the ends? Oncogene 23, 72837289.
Harrison, D.E., 1979a. Mouse erythropoietic stem cell lines function normally 100 months: loss
related to number of transplantations. Mech. Ageing and Devel. 9, 427-433.
Harrison, D.E., 1979b. Proliferative capacity of erythropoietic stem cell lines and aging: an overview.
Mech. Ageing and Devel. 9, 409-426.
Harrison, D.E., Jordan, C.T., Zhong, R.K., et al., 1993. Primitive hemopoietic stem cells: direct assay
of most productive populations by competitive repopulation with simple binomial, correlationand covariance calculations. Exp. Hematol. 21, 206-219.
Harrison, J.D., Muirhead, C.R., 2003. Quantitative comparisons of cancer induction in humans byinternally deposited radionuclides and external radiation. Int. J. Radiat. Biol. 79, 1-13.
Hartmann, C., 2006. A Wnt canon orchestrating osteoblastogenesis. Trends Cell Biol. 16, 151-158.
Hayashita, Y., Osada, H., Tatematsu, Y., et al., 2005. A polycistronic microRNA cluster, miR-17-92,
is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res. 65, 96289632.
He, L., He, X., Lim, L.P., et al., 2007. A microRNA component of the p53 tumour suppressor
network. Nature 447, 1130-1134.
Heenen, M., Galand, P., 1997. The growth fraction of normal human epidermis. Dermatol. 194, 313317.
Hei, T.K., Piao, C.Q., Sutter, T., et al., 1996. Cellular and molecular alterations in human epithelial
cells transformed by high LET radiation. Adv. Space Res. 18, 137-148.

242

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10231	
  10232	
  10233	
  10234	
  10235	
  10236	
  10237	
  10238	  10239	
  10240	
  10241	
  10242	
  10243	
  10244	
  10245	
  10246	
  10247	  10248	  10249	
  10250	
  10251	
  10252	
  10253	
  10254	
  10255	
  10256	
  10257	
  10258	  10259	
  10260	
  10261	
  10262	
  10263	
  10264	
  10265	
  10266	  10267	  10268	  10269	
  10270	  10271	
  10272	
  10273	  10274	
  10275	  10276	  10277	  10278	  10279	
  10280	  10281	  10282	  10283	  Hei, T.K., Piao, C.Q., Wu, L.J., et al., 1998. Genomic instability and tumorigenic induction in
immortalized human bronchial epithelial cells by heavy ions. Adv. Space Res. 22, 1699-1707.
Heidenreich, W.F., Jacob, P., Paretzke, H.G., et al., 1999. Two-step model for the risk of fatal andincidental lung tumors in rats exposed to radon. Radiat. Res. 151, 209-217.
Hemmati, H.D., Nakano, I., Lazareff, J.A., et al., 2003. Cancerous stem cells can arise from pediatricbrain tumors. Proc. Natl. Acad. Sci. U S A. 100, 15178-15183.
Henderson, T.O., Amsterdam, A., Bhatia, S., et al., 2010. Systematic review: surveillance for breast
cancer in women treated with chest radiation for childhood, adolescent, or young adult cancer.
Ann. Intern. Med. 152, 444-455.
Hendry, J.H., 1985. Survival curves for normal-tissue clonogens: a comparison of assessments using
in vitro, transplantation, or in situ techniques. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
47, 3-16.
Hendry, J.H., 2002. Radiation-induced apoptosis and its role in tissue response. Int. Congr. Series
1236, 415-421.
Hendry, J.H., Cai, W.B., Roberts, S.A., et al., 1997. p53 deficiency sensitizes clonogenic cells toirradiation in the large but not the small intestine. Radiat. Res. 148, 254-259.
Hendry, J.H., Edmundson, J.M., Potten, C.S., 1980. The radiosensitivity of hair follicles in mousedorsum and tail. Radiat. Res. 84, 87-96.
Hendry, J.H., Lajtha, L.G., 1975. Response of mouse bone marrow to low dose rates, split acute doses,
and multiple daily fractions. Proc. 6th Gray Conf. London. John Wiley, London, UK, pp. 308312.
Hendry, J.H., Potten, C.S., 1982. Intestinal cell radiosensitivity: a comparison for cell death assayedby apoptosis or by a loss of clonogenicity. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med.
42, 621-628.
Hendry, J.H., Potten, C.S., Chadwick, C., et al., 1982. Cell death (apoptosis) in the mouse small
intestine after low doses: effects of dose-rate, 14.7 MeV neutrons, and 600 MeV (maximum
energy) neutrons. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 42, 611-620.
Hendry, J.H., Yang, J., 1995. The response of bone marrow to low LET irradiation. In: Radiation
Toxicology - Bone Marrow and Leukaemia. Eds: J.H. Hendry & B.I. Lord. Taylor & Francis
London, UK, pp. 91-116.
Henrichs, K., Bogner, L., Nekolla, E., et al., 1995. Extended dosimetry for studies with Ra-224
patients. In: van Kaick, G., Karaoglou, A., Kellerer, A.M. (Eds.), Health Effects of InternallyDeposited Radionuclides: Emphasis on Radium and Thorium. World Scientific, Singapore, pp.
33-38.
Henschler, R., Brugger, W., Luft, T., et al., 1994. Maintenance of transplantation potential in ex vivo
expanded CD34(+)-selected human peripheral blood progenitor cells. Blood 84, 2898-2903.
Herbst, R.S., Heymach, J.V., Lippman, S.M., 2008. Molecular origins of lung cancer. N. Engl. J. Med.
359, 1367-1380.
Hill, D.A., Preston-Martin, S., Ross, R.K., et al., 2002. Medical radiation, family history of cancer,
and benign breast disease in relation to breast cancer risk in young women, USA. Cancer
Causes & Control 13, 711-718.
Hiraoka, T., Miller, R.C., Gould, M.N., et al., 1985. Survival of human thyroid cells after x-ray
irradiation. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 47, 299-307.
Hirouchi, T., Akabane, M., Tanaka, S., et al., 2011. Cell surface marker phenotypes and gene
expression profiles of murine radiation-induced acute myeloid leukemia stem cells are similar
to those of common myeloid progenitors. Radiat. Res. 176, 311-322.
Hirsch, F.R., Varella-Garcia, M., Bunn, P.A.J., et al., 2003. Epidermal growth factor receptor in nonsmall-cell lung carcinomas: correlation between gene copy number and protein expression andimpact on prognosis. J. Clin. Oncol. 21, 3798-3807.
Hiyama, K., Hirai, Y., Kyoizumi, S., et al., 1995a. Activation of telomerase in human lymphocytes
and hematopoietic progenitor cells. J. Immunol. 155, 3711-3715.
Hiyama, K., Hiyama, E., Ishioka, S., et al., 1995b. Telomerase activity in small-cell and non-smallcell lung cancers. J. Natl. Cancer Inst. 87, 895-902.

243

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10284	  10285	
  10286	  10287	  10288	  10289	
  10290	  10291	  10292	  10293	  10294	  10295	  
10296	  10297	  10298	
  10299	
  10300	
  10301	
  10302	
  10303	
  10304	
  10305	
  
10306	
  10307	
  10308	
  10309	
  10310	
  10311	
  10312	
  10313	
  10314	
  10315	
  10316	
  10317	  10318	  10319	
  10320	
  10321	
  10322	
  10323	
  10324	
  10325	
  10326	
  10327	  10328	  10329	
  10330	
  10331	
  10332	
  10333	
  10334	
  10335	
  Hoffman, D.A., Lonstein, J.E., Morin, M.M., et al., 1989. Breast cancer in women with scoliosis
exposed to multiple diagnostic rays. J. Natl. Cancer Inst. 81, 1307-1312.
Hong, K.U., Reynolds, S.D., Giangreco, A., et al., 2001. Clara cell secretory protein-expressing cellsof the airway neuroepithelial body microenvironment include a label-retaining subset and are
critical for epithelial renewal after progenitor cell depletion. Am. J. Respir. Cell. Mol. Biol. 24,
671-681.
Hong, K.U., Reynolds, S.D., Watkins, S., et al., 2004. In vivo differentiation potential of tracheal
basal cells: evidence for multipotent and unipotent subpopulations. Am. J. Physiol. Lung Cell
Mol. Physiol. 286, L643-649.
Horwitz, E.M., Le Blanc, K., Dominici, M., et al., 2005. Clarification of the nomenclature for MSC:
The International Society for Cellular Therapy position statement. Cytotherapy 7, 393-395.
Houghton, J., Stoicov, C., Nomura, S., et al., 2004. Gastric cancer originating from bone marrowderived cells. Science 306, 1568-1571.
Howe, G.R., 1995. Lung cancer mortality between 1950 and 1987 after exposure to fractionatedmoderate-dose-rate ionizing radiation in the Canadian fluoroscopy cohort study and acomparison with lung cancer mortality in the Atomic Bomb survivors study. Radiat Res. 142,
295-304.
Howe, G.R., McLaughlin, J., 1996. Breast cancer mortality between 1950 and 1987 after exposure to
fractionated moderate-dose-rate ionizing radiation in the Canadian fluoroscopy cohort study
and a comparison with breast cancer mortality in the atomic bomb survivor study. Radiat. Res.
145, 694-707.
Howlett, A.R., Carter, V.C., Martin, G.S., et al., 1988. pp60v-src tyrosine kinase is expressed andactive in sarcoma-free avian embryos microinjected with Rous sarcoma virus. Proc. Natl. Acad.
Sci. U S A, 85, 7587-7591.
Hsu, P.H., Hanawalt, P.C., Nouspikel, T., 2007. Nucleotide excision repair phenotype of human acute
myeloid leukemia cell lines at various stages of differentiation. Mut. Res. 614, 3-15.
Hsu W.L., Preston D.L., Soda M., et al., 2013. The incidence of leukemia, lymphoma and multiple
myeloma among atomic bomb survivors: 1950-2001. Radiat. Res. 179, 361-382.
Hu, M., Krause, D., Greaves, M., et al., 1997. Multilineage gene expression precedes commitment in
the hemopoietic system. Genes Dev. 11, 774-785.
Hua, G., Thin, T.H., Feldman, R., et al., 2012. Crypt base columnar stem cells in small intestines ofmice are radioresistant. Gastroenterology 143, 1266-1276.
Huelsken J., B.J., 2002. The Wnt signaling pathway. J. Cell. Sci. 115, 3977-3978.
Hulse, E.V., Lewkowicz, S.J., Batchelor, A.L., et al., 1983. Incidence of radiation-induced skintumours in mice and variations with dose rate. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem.
Med. 44, 197-206.
Humblet, C., Defresne, M.P., Greimers, R., et al., 1989. Further studies on the mechanism of radiationinduced thymic lymphoma prevention by bone marrow transplantation in C57BL mice.
Leukemia. 3, 813-818.
Humblet, C., Greimers, R., Boniver, J., et al., 1997. Stages in the development of radiation-induced
thymic lymphomas in C57 BL/Ka mice: preleukemic cells become progressively resistant to thetumor preventing effects of a bone marrow graft. Exp. Hematol. 25, 109-113.
Hume, W.J., Potten, C.S., 1979. Advances in epithelial kinetics--an oral view. J. Oral Pathol. 8, 3-22.
Humphreys, E.R., Isaacs, K.R., Raine, T.A., et al., 1993. Myeloid leukaemia and osteosarcoma in
CBA/H mice given 224Ra. Int. J. Radiat. Biol. 64, 231-235.
Humphreys, E.R., Loutit, J.F., Stones, V.A., 1987. The induction by 239Pu of myeloid leukaemia andosteosarcoma in female CBA mice. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 51, 331339.
Humphries, A., Wright, N.A., 2008. Colonic crypt organization and tumorigenesis. Nat. Rev. Cancer
8, 415-424.
Huss, R., 2000. Perspectives on the morphology and biology of CD34-negative stem cells. J
Hematother. Stem Cell Res. 9, 783-793.

244

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10336	
  10337	
  10338	  10339	
  10340	
  10341	
  10342	
  10343	
  10344	
  10345	
  10346	
  10347	  10348	  10349	
  10350	
  10351	
  10352	
  10353	
  10354	
  10355	
  10356	
  10357	
  10358	  10359	
  10360	
  10361	
  10362	
  10363	  10364	
  10365	
  10366	
  10367	  10368	  10369	
  10370	
  10371	
  10372	
  10373	
  10374	
  10375	
  10376	
  10377	  10378	  10379	
  10380	  10381	  10382	  10383	  10384	  10385	
  10386	  10387	  Huvos, A.G., 1991. Bone tumors: diagnosis, treatment and prognosis, 2nd edition. WB Saunders,Philadelphia.
Hyodo, M., Sadamoto, A., Hinohira, Y., et al., 1999. Tongue cancer as a complication of dyskeratosis
congenita in a woman. Am. J. Otolaryngol. 20, 405-407.
ICRP, 1975. Report of the Task Group on Reference Man. ICRP Publication, Pergamon Press, Oxford,
UK, pp. 1-480.
ICRP, 1991. 1990 Recommendations of the International Commission on Radiological Protection.
ICRP Publication 60. Ann. ICRP 21, 1-3.
ICRP, 1992. The biological basis for dose limitation in the skin. ICRP Publication 59. Ann. ICRP 22,
2.
ICRP, 1993. Protection against Radon-222 at home and work: ICRP Publication 65. Ann. ICRP 23 (2).
ICRP, 1995. Basic anatomical and physiological data for use in radiological protection: The skeleton.
ICRP Publication 70. Ann. ICRP 25, (2).
ICRP, 1998. Genetic susceptibility to cancer. ICRP Publication 79. Ann. ICRP 28 (1-2).
ICRP, 2000. Avoidance of radiation injuries from medical interventional procedures. ICRP
Publication 85. Ann. ICRP 30, (2).
ICRP, 2002. Basic anatomical and physiological data for use in radiological protection: reference
values. ICRP Publication 89. Ann. ICRP 32 (3-4).
ICRP, 2003. Biological effects after prenatal irradiation (embryo and fetus). ICRP Publication 90.
Ann. ICRP 33 (1-2).
ICRP, 2005. Low-dose extrapolation of radiation-related cancer risk. ICRP Publication 99. Ann.
ICRP 35 (4).
ICRP, 2006. Human alimentary tract model for radiological protection. ICRP Publication 100. Ann.
ICRP 36 (1-2).
ICRP, 2007. The 2007 Recommendations of the International Commission on Radiological Protection.
ICRP Publication 103. Ann. ICRP 37 (2-4).
ICRP, 2010. Lung cancer risk from radon and progeny and statement on radon. ICRP Publication 115.
Ann. ICRP 40 (1).
ICRP, 2012. ICRP statement on tissue reactions, and Early and late effects of radiation in normal
tissues and organs--threshold doses for tissue reactions in a radiation protection context. ICRP
Publication 118. Ann. ICRP 41 (1/2).
Ihle, J.N., Joseph, D.R., Pazmino, N.H., 1976. Radiation leukemia in C57BL/6 mice. II. Lack of
ecotropic virus expression in the majority of lymphomas. J. Exp. Med. 144, 1406-1423.
Ijiri K., Potten, C.S., 1984. The re-establishment of hypersensitive cells in the crypts of irradiatedmouse intestine. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 46, 609-623.
Ikeda, S., Hachisu, R., Yamaguchi, A., et al., 2000. Radiation retards muscle differentiation but does
not affect osteoblastic differentiation induced by bone morphogenetic protein-2 in C2C12
myoblasts. Int. J. Radiat. Biol. 76, 403-411.
Imai, Y., Nakao, I., 1987. In vivo radiosensitivity and recovery pattern of the hematopoietic precursor
cells and stem cells in mouse bone marrow. Exp. Hematol. 15, 890-895.
Imaoka, T., Nishimura, M., Iizuka, D., et al., 2011. Pre- and postpubertal irradiation induces
mammary cancers with distinct expression of hormone receptors, ErbB ligands, anddevelopmental genes in rats. Mol. Carcinog. 50, 539-552.
Imaoka, T., Nishimura, M., Kakinuma, S., et al., 2007. High Relative Biologic Effectiveness ofCarbon Ion Radiation on Induction of Rat Mammary Carcinoma and its Lack of H-ras andTp53 mutations. Int. J. Radiat. Oncol. Biol. Phys. 69, 194-203.
Inano, H., Suzuki, K., Onoda, M., et al., 1996. Susceptibility of fetal, virgin, pregnant and lactatingrats for the induction of mammary tumors by gamma rays. Radiat. Res. 145, 708-713.
Inomata, K., Aoto, T., Binh, N.T., 2009. Genotoxic stress abrogates renewal of melanocyte stem cellsby triggering their differentiation. Cell 137, 1088-1099.
Inoue, K., Ohgushi, H., Yoshikawa, T., et al., 1997. The effect of aging on bone formation in poroushydroxyapatite: biochemical and histological analysis. J. Bone Mineral Res. 12, 989-994.

245

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10388	  10389	
  10390	  10391	
  
10392	
  10393	  10394	  10395	  
10396	  10397	  10398	
  10399	
  10400	
  10401	
  
10402	
  
10403	
  
10404	
  
10405	
  
10406	
  
10407	
  
10408	
  
10409	
  10410	
  10411	
  10412	
  10413	
  10414	
  10415	
  
10416	
  10417	
  10418	  10419	
  10420	
  10421	
  
10422	
  
10423	
  10424	
  10425	
  
10426	
  10427	
  10428	  10429	
  10430	
  10431	
  
10432	
  10433	
  10434	
  10435	
  
10436	
  10437	
  10438	
  10439	
  Inoue, T., Hirabayashi, Y., Mitsui, H., et al., 1995. Survival of spleen colony-forming units (CFU-S)of irradiated bone marrow cells in mice: evidence for the existence of a radioresistantsubfraction. Exp. Hematol. 23, 1296-1300.
Inskip, P.D., Monson, R.R., Wagoner, J.K., et al., 1990. Cancer mortality following radium treatment
for uterine bleeding. Radiat. Res. 123, 331-344.
Iscove, N.N., Nawa, K., 1997. Hematopoietic stem cells expand during serial transplantation in vivo
without apparent exhaustion. Current Biol. 7, 805-808.
Ishitani, T., Kishida, S., Hyodo-Miura, J., et al., 2003. The TAK1-NLK mitogen-activated protein
kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-cateninsignaling. Mol. Cell. Biol. 23, 131-139.
Ito, H., Meistrich, M.L., Barkley, H.T., Jr., et al., 1986. Protection of acute and late radiation damageof the gastrointestinal tract by WR-2721. Int. J. Radiat. Oncol. Biol. Phys. 12, 211-219.
Ito, K., Hirao, A., Arai, F., et al., 2004. Regulation of oxidative stress by ATM is required for selfrenewal of haematopoietic stem cells. Nature 431, 997-1002.
Ito, K., Hirao, A., Arai, F., et al., 2006. Reactive oxygen species act through p38 MAPK to limit thelifespan of hematopoietic stem cells. Nat. Med. 12, 446-451.
Ito, M., Cotsarelis, G., 2008. Is the hair follicle necessary for normal wound healing? J. Invest.
Dermatol. 128, 1059-1061.
Ito, M., Liu, Y., Yang, Z., et al., 2005. Stem cells in the hair follicle bulge contribute to wound repair
but not to homeostasis of the epidermis. Nat. Med. 11, 1351-1354.
Ito, T., Seyama, T., Iwamoto, K.S., et al., 1993a. In vitro irradiation is able to cause RET oncogene
rearrangement. Cancer Res. 53, 2940-2943.
Ito, T., Seyama, T., Mizuno, T., et al., 1993b. Induction of BCR-ABL fusion genes by in vitro Xirradiation. Jpn. J. Cancer Res. 84, 105-109.
Ivanovic, Z., Bartolozzi, B., Bernabei, P.A., et al., 2000. Incubation of murine bone marrow cells inhypoxia ensures the maintenance of marrow-repopulating ability together with the expansion ofcommitted progenitors. Br. J. Haematol. 108, 424-429.
Iwanaga, M., Hsu, W.L., Soda, M., et al., 2011. Risk of myelodysplastic syndromes in people exposed
to ionizing radiation: a retrospective cohort study of Nagasaki atomic bomb survivors. J. Clin.
Oncol. 29, 428-434.
Jacob, P., Rühm, W., Walsh, L., et al., 2009. Is cancer risk of radiation workers larger than expected.
Occup. Environ. Med. 66, 789-796.
Jaks, V., Barker, N., Kasper, M., et al., 2008. Lgr5 marks cycling, yet long-lived, hair follicle stem
cells. Nat. Genet. 40, 1291-1299.
Janowski, M., Borremans, B., Hooghe, R., et al., 1987. Search for genes involved in murine thymic
lymphomagenesis, Proc. Eighth International Congress of Radiation Research. Taylor &Francis, London, pp. 482-487.
Japon, M.A., Rubinstein, M., Low, M.J., 1994. In situ hybridization analysis of anterior pituitaryhormone gene expression during fetal mouse development. J. Histochem. Cytochem. 42, 11171125.
Jawad, M., Giotopoulos, G., Cole, C., et al., 2007. Target cell frequency is a genetically determinedrisk factor in radiation leukaemogenesis. Br. J. Radiol. 80, S56-62.
Jawad, M., Seedhouse, C.H., Russell, N., et al., 2006. Polymorphisms in human homeobox HLX1 andDNA repair RAD51 genes increase the risk of therapy-related acute myeloid leukemia. Blood
108, 3916-3918.
Jeanmart, M., Lantuejoul, S., Fievet, F., et al., 2003. Value of immunohistochemical markers inpreinvasive bronchial lesions in risk assessment of lung cancer. Clin. Cancer Res. 9, 2195-2203.
Jensen, E.V., Cheng, G., Palmieri, C., et al., 2001. Estrogen receptors and proliferation markers inprimary and recurrent breast cancer. Proc. Natl. Acad. Sci. U.S.A. 98, 15197-15202.
Jensen, S.M., Meijer, S.L., Kurt, R.A., et al., 2003. Regression of a mammary adenocarcinoma in
STAT6-/- mice is dependent on the presence of STAT6-reactive T cells. J. Immunol. 170,
2014-2021.

246

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10440	
  10441	
  10442	
  10443	
  10444	
  10445	
  
10446	
  10447	
  10448	  10449	
  10450	
  10451	
  10452	
  10453	
  10454	
  10455	
  10456	
  10457	
  10458	  10459	
  10460	
  10461	
  
10462	
  
10463	  
10464	  
10465	
  10466	  
10467	  
10468	  
10469	
  10470	  10471	
  10472	
  10473	  10474	  10475	  
10476	  10477	  10478	  10479	
  10480	  10481	
  
10482	
  10483	  10484	  10485	
  10486	  10487	  
10488	  10489	
  10490	
  
10491	
  Jensen, U.B., Lowell, S., Watt, F.M., 1999. The spatial relationship between stem cells and theirprogeny in the basal layer of human epidermis: a new view based on whole-mount labelling
and lineage analysis. Development 126, 2409-2418.
Ji, H., Ramsey, M.R., Hayes, D.N., et al., 2007. LKB1 modulates lung cancer differentiation andmetastasis. Nature 448, 807-810.
Jiang, X.R., Jimenez, G., Chang, E., et al., 1999. Telomerase expression in human somatic cells does
not induce changes associated with a transformed phenotype. Nat. Genet. 21, 111-114.
Jiang, Y., Lenvik, T., Blackstad, M., et al., 2002. Multipotent progenitor cells can be isolated frompostnatal murine bone marrow, muscle, and brain. Exp. Hemtal. 30, 896-904.
Jilka, R.L., Weinstein, R.S., Parfitt, A.M., et al., 2007. Quantifying osteoblast and osteocyte
apoptosis: challenges and rewards. J. Bone Miner. Res. 22, 1492-1501.
Jin, Y.W., Na, Y.J., Lee, Y.J., et al., 2008. Comprehensive analysis of time- and dose-dependent
patterns of gene expression in a human mesenchymal stem cell line exposed to low-dose
ionizing radiation. Oncol. Rep. 19, 135-144.
Jirtle, R.L., Michalopoulos, G., 1985. A clonal assay in vivo for parenchymal heptocytes. In: Potten,
C.S., Hendry, J.H. (Eds.), Cell Clones: Manual of Mammalian Cell Techniques. Churchill
Livingstone, Edinburgh, pp. 139-151.
Johnson, L., Mercer, K., Greenbaum, D., et al., 2001. Somatic activation of the K-ras oncogene causes
early onset lung cancer in mice. Nature 410, 1111-1116.
Jonker, J.W., Freeman, J., Bolscher, E., et al., 2005. Contribution of the ABC transporters Bcrp1 andMdr1a/1b to the side population phenotype in mammary gland and bone marrow of mice. StemCells 23, 1059-1065.
Joshi, P.A., Jackson, H.W., Beristain, A.G., et al., 2010. Progesterone induces adult mammary stem
cell expansion. Nature 465, 803-807.
Jung, P., Sato, T., Merlos-Suarez, A., et al., 2011. Isolation and in vitro expansion of human colonicstem cells. Nature Med. 17, 1225-1227.
K., I., Potten, C.S., 1984. The re-establishment of hypersensitive cells in the crypts of irradiatedmouse intestine. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 46, 609-623.
Kabat, G.C., Wynder, E.L., 1984. Lung cancer in nonsmokers. Cancer 53, 1214-1221.
Kadhim, M.A., Lorimore, S.A., Hepburn, M.D., et al., 1994. Alpha-particle-induced chromosomal
instability in human bone marrow cells. Lancet 344, 987-988.
Kadhim, M.A., Macdonald, D.A., Goodhead, D.T., et al., 1992. Transmission of chromosomal
instability after plutonium alpha-particle irradiation. Nature 355, 738-740.
Kadhim, M.A., Wright, E.G., 1998. Radiation-induced transmissable chromosomal instability inhaemopoietic stem cells. Adv. Space Res. 22, 587-596.
Kajstura, J., Rota, M., Hall, S.R., et al., 2011. Evidence for human lung stem cells. N. Engl. J. Med.
364, 1795-1806.
Kameda, Y., Nishimaki, T., Miura, M., et al., 2007. Mash1 regulates the development of C cells inmouse thyroid glands. Dev. Dyn. 236, 262-270.
Kamiya, K., Gould, M.N., Clifton, K.H., 1990. Differential control of alveolar and ductal
development in grafts of monodispersed rat mammary epithelium. Expt. Biol. Med. 196, 284292.
Kamiya, K., Gould, M.N., Clifton, K.H., 1998. Quantitative studies of ductal versus alveolar
differentiation from rat mammary clonogens. Proc. Soc. Exp. Biol. Med. 219, 217-225.
Kamiya, K., Higgins, P.D., Tanner, M.A., et al., 1999. Kinetics of mammary clonogenic cells and rat
mammary cancer induction by X-rays or fission neutrons. J. Radiat. Res. 40, 128-137.
Kamiya, K., Kim, N.D., Gould, M.N., et al., 1991. Repair of potentially lethal damage in rat
mammary clonogens following irradiation in organoid culture. Int. J. Radiat. Biol. 59, 12071216.
Kamiya, K., Yasukawa-Barnes, J., Mitchen, J.M., et al., 1995. Evidence that carcinogenesis involves
an imbalance between epigenetic high-frequency initiation and suppression of promotion. Proc.
Natl. Acad. Sci. U S A. 92, 1332-1336.

247

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10492	
  
10493	  
10494	
  
10495	  
10496	  
10497	  
10498	
  10499	
  10500	
  10501	
  
10502	
  10503	
  10504	
  10505	
  
10506	
  10507	
  10508	
  10509	
  10510	
  10511	
  10512	
  10513	
  10514	
  10515	
  10516	
  10517	
  10518	
  10519	
  10520	
  10521	
  10522	
  10523	
  10524	
  10525	
  
10526	
  10527	
  10528	
  10529	
  10530	
  10531	
  10532	
  10533	
  10534	
  10535	
  
10536	
  10537	
  10538	
  10539	
  10540	
  10541	
  10542	
  10543	
  10544	
  Kanitakis, J., Bourchany, D., Faure, M., et al., 1999. Expression of the hair stem cell-specific keratin
15 in pilar tumors of the skin. Eur. J. Dermatol. 9, 363-365.
Kaplan, H.S., 1974. Leukemia and lymphoma in experimental and domestic animals. Ser. Haematol. 7,
94-163.
Kaplan, H.S., Carnes, W.H., Brown, M.B., et al., 1956. Indirect induction of lymphomas in irradiatedmice: I. Tumor incidence and morphology in mice bearing nonirradiated thymic grafts. Cancer
Res. 16, 422-425.
Karagas, M.R., Nelson, H.H., Zens, M.S., et al., 2007. Squamous cell and basal cell carcinoma of theskin in relation to radiation therapy and potential modification of risk by sun exposure.
Epidemiology 18, 776-784.
Karam, S.M., Straiton, T., Hassan, W.M., et al., 2003. Defining epithelial cell progenitors in thehuman oxyntic mucosa. Stem Cells 21, 322-336.
Karaoglu, A.O., Kadikoylu, G., Yukselen, V., et al., 2004. Gastrointestinal lesions and Helicobacter
pylori in patients with myeloproliferative disorders. Saudi Med. J. 25, 1913-1916.
Karlseder, J., Broccoli, D., Dai, Y., et al., 1999. p53- and ATM-dependent apoptosis induced bytelomeres lacking TRF2. Science 283, 1321-1325.
Kassis, A.I., 2004. In vivo validation of the bystander effect. Human & Exp.Toxicol. 23, 71-73.
Kataoka, Y., Perrin, J., Grdina, D.J., 1993. Induction of hprt mutations in mice after exposure to
fission-spectrum neutrons or 60Co gamma rays. Radiat Res. 136, 289-292.
Katoh, R., Bray, C.E., Suzuki, K., et al., 1995. Growth activity in hyperplastic and neo plastis human
thyroid determined by an immunohistochemical staining procedure using monoclonal antibody.
Hum. Pathol. 26, 139-146.
Katz, F., Reeves, B.R., Alexander, S., et al., 1993 Leukaemia arising in donor cells following
allogeneic bone marrow transplantation for beta thalassaemia demonstrated by immunological,
DNA and molecular cytogenetic analysis. Br. J. Haematol. 85, 326-331.
Kauffman, S.L., 1980. Cell proliferation in the mammalian lung. Int. Rev. Exp. Pathol. 22, 131-191.
Kaur, P., 2006. Interfollicular epidermal stem cells: identification, challenges, potential. J. Invest.
Dermatol. 126, 1450-1458.
Kawai, H., Ishii, A., Washiya, K., et al., 2005. Estrogen receptor alpha and beta are prognostic factors
in non-small cell lung cancer. Clin. Cancer Res. 11, 5084-5089.
Kay, H.E., 1965. How many cell-generations? Lancet 11, 418-419.
Kayahara, T., Sawada, M., Takaishi, S., et al., 2003. Candidate markers for stem and early progenitor
cells, Musashi-1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine.
FEBS letters 535, 131-135.
Keating, A., 2012. Mesenchymal stromal cells: new directions. Cell Stem Cell 10, 709-716.
Kelemen, E., Calvo, W., 1982. Prenatal hematopoiesis in human bone marrow and its developmental
antecedents, The Human Bone Marrow: anatomy, physiology, and pathophysiology. CRC Press
Inc, Boca Raton, FL, USA, pp. 3-41.
Kellerer, A.M., Rossi, H.M., 1972. The theory of dual radiation action. Curr. Topics Radiat. Res.
Quart. 8, 85-158.
Kelly, G., Stegelmeier, B.L., Hahn, F.F., 1995. p53 alterations in plutonium-induced F344 rat lungtumors. Radiat. Res. 142, 263-269.
Kennedy, A.R., Fox, M., Murphy, G., et al., 1980. Relationship between x-ray exposure andmalignant transformation in C3H 10T1/2 cells. Proc. Natl. Acad. Sci. U S A. 77, 7262-7266.
Kennedy, A.R., Little, J.B., 1978. Protease inhibitors suppress radiation-induced malignant
transformation in vitro. Nature 276, 825-826.
Kenney, N.J., Smith, G.H., Lawrence, E., et al., 2001. Identification of stem cell units in the terminalend bud and duct of the mouse mammary gland. J. Biomed. and Biotechnol. 1, 133-143.
Khan, S.A., Rogers, M.A., Obando, J.A., et al., 1994. Estrogen receptor expression of benign breastepithelium and its association with breast cancer. Cancer Res. 54, 993-997.
Khavari, P.A., 2006. Modelling cancer in human skin tissue. Nat. Rev. Cancer 6, 270-280.
Khosla, S., Westendorf, J.J., Modder, U.I., 2010. Concise review: Insights from normal bone
remodeling and stem cell-based therapies for bone repair. Stem Cells 28, 2124-2128.

248

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10545	
  
10546	
  10547	
  10548	  10549	
  10550	
  10551	
  10552	
  10553	
  10554	
  10555	
  10556	
  10557	
  10558	
  10559	
  10560	
  10561	
  
10562	
  
10563	
  
10564	
  
10565	
  10566	
  
10567	  
10568	  
10569	
  10570	
  10571	
  10572	
  10573	
  10574	
  10575	
  
10576	
  10577	  10578	  10579	
  10580	
  10581	
  
10582	
  
10583	  
10584	  
10585	
  10586	  
10587	  
10588	  
10589	
  10590	
  
10591	
  
10592	
  
10593	
  
10594	
  
10595	  
10596	
  Kiel, M.J., He, S., Ashkenazi, R., et al., 2007. Haematopoietic stem cells do not asymmetricallysegregate chromosomes or retain BrdU. Nature 449, 238-242.
Kiel, M.J., Yilmaz, O.H., Iwashita, T., et al., 2005. SLAM family receptors distinguish hematopoietic
stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121, 1109-1121.
Kim, C.F., Jackson, E.L., Woolfenden, A.E., et al., 2005. Identification of bronchioalveolar stem cells
in normal lung and lung cancer. Cell 121, 823-835.
Kim, N.D., Oberley, T.D., Yasukawa-Barnes, J., et al., 2000. Stem cell characteristics of transplanted
rat mammary clonogens. Exp. Cell Res. 260, 146-159.
Kinzler, K.W., Vogelstein, B., 1996. Lessons from hereditary colorectal cancer. Cell 87, 159-170.
Kishikawa, M., Koyama, K., Iseki, M., et al., 2005. Histologic characteristics of skin cancer in
Hiroshima and Nagasaki: Background incidence and radiation effects. International Journal ofCancer 117, 363-369.
Kleber, M., Sommer, L., 2004. Wnt signaling and the regulation of stem cell function. Curr. Opin.
Cell Biol. 16, 681-687.
Kleeberger, W., Versmold, A., Rothamel, T., et al., 2003. Increased chimerism of bronchial andalveolar epithelium in human lung allografts undergoing chronic injury. Am. J. Pathol. 162,
1487-1494.
Klein, A.M., Nakagawa, T., Ichikawa, R., et al., 2010. Mouse germ line stem cells undergo rapid andstochastic turnover. Cell Stem Cell 7, 214-224.
Kleinerman, R.A., 2009. Radiation-sensitive genetically susceptible pediatric sub-populations. Pediatr.
Radiol. 39 Suppl 1, S27-31.
Klihues, P., Cavenee, K., 2000. World Health Organization Classification of Tumors of the Nervous
System. IARC Press, Lyon, France.
Klugbauer, S., Lengfelder, E., Demidchik, E.P., et al., 1995. High prevalence of RET rearrangementin thyroid tumors of children from Belarus after the Chernobyl reactor accident. Oncogene 11,2459-2467.
Knight, D.A., Holgate, S.T., 2003. The airway epithelium: structural and functional properties inhealth and disease. Respirology 8, 432-446.
Knothe Tate, M.L., Adamson, J.R., Tami, A.E., et al., 2004. The osteocyte. Int. J. Biochem. & Cell
Biol. 36, 1-8.
Knudson, A.G., Jr., 1971. Mutation and cancer: statistical study of retinoblastoma. Proc. Natl. Acad.
Sci. U S A. 68, 820-823.
Ko, Y.C., Lee, C.H., Chen, M.J., et al., 1997. Risk factors for primary lung cancer among nonsmoking women in Taiwan. Int. J. Epidemiol. 26, 24-31.
Kodama, K., Ozasa, K., Okubo, T., 2012. Radiation and cancer risk in atomic-bomb survivors. J.
Radiol. Prot. 32, N51-54.
Kogai, T., Endo, T., Saito, T., et al., 1997. Regulation by thyroid-stimulating hormone of
sodium/iodide symporter gene expression and protein levels in FRTL-5 cells. Endocrinol. 138,
2227-2232.
Kolf, C.M., Cho E., Tuan R.S., 2007. Biology of adult mesenchymal stem cells: regulation of niche,
self-renewal and differentiation. Arthritis Res. and Therapy 9, 204.
Kominami, R., Niwa, O., 2006. Radiation carcinogenesis in mouse thymic lymphomas. Cancer Sci.
97, 575-581.
Kondo, H., Searby, N.D., Mojarrab, R., et al., 2009. Total-body irradiation of postpubertal mice with(137)Cs acutely compromises the microarchitecture of cancellous bone and increases
osteoclasts. Radiat. Res. 171, 283-289.
Kordon, E.C., Smith, G.H., 1998. An entire functional mammary gland may comprise the progeny
from a single cell. Development 125, 1921-1930.
Kosaka, T., Yatabe, Y., Endoh, H., et al., 2004. Mutations of the epidermal growth factor receptor
gene in lung cancer: biological and clinical implications. Cancer Res. 64, 8919-8923.
Koshurnikova, N.A., Gilbert, E.S., Sokolnikov, M., et al., 2000. Bone cancers in Mayak workers.
Radiat. Res. 154, 237-245.

249

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10597	  
10598	
  10599	
  10600	
  10601	
  
10602	
  
10603	  
10604	  10605	  
10606	  10607	  10608	  10609	
  10610	
  10611	
  10612	
  10613	  10614	  10615	  
10616	  10617	  10618	  10619	
  10620	
  10621	
  10622	
  10623	  10624	  10625	  
10626	
  10627	  10628	  10629	
  10630	
  10631	
  10632	
  10633	
  10634	  10635	  
10636	  10637	  10638	  10639	
  10640	  10641	
  10642	
  10643	  10644	  10645	  
10646	  10647	
  Kothari, M.S., Ali, S., Buluwela, L., et al., 2003. Purified malignant mammary epithelial cells
maintain hormone responsiveness in culture. Br. J. Cancer 88, 1071-1076.
Koyi, H., Hillerdal, G., Branden, E., 2002. A prospective study of a total material of lung cancer froma county in Sweden 1997-1999: gender, symptoms, type, stage, and smoking habits. Lung
Cancer 36, 9-14.
Krane, S.M., 2005. Identifying genes that regulate bone remodeling as potential therapeutic targets. J.
Exp. Med. 201, 841-843.
Kratochwil, K., 1969. Organ specificity in mesenchymal induction demonstrated in the embryonicdevelopment of the mammary gland of the mouse. Dev. Biol. 20, 46-71.
Krause, D.S., 2002. Regulation of hematopoietic stem cell fate. Oncogene 21, 3262-3269.
Kreja, L., Thoma, S., Selig, C., et al., 1993. The effect of recombinant human stem cell factor andbasic fibroblast growth factor on the in vitro radiosensitivity of CD34+ hematopoietic
progenitors from human umbilical cord blood. Exp. Hematol. 21, 1436-1441.
Krestinina, L., Preston, D.L., Davis, F.G., et al., 2010 Leukemia incidence among people exposed tochronic radiation from the contaminated Techa River, 1953-2005. Radiat. Environ. Biophys. 49,
195-201.
Krestinina, L.Y., Preston, D.L., Ostroumova, E.V., et al., 2005 Protracted radiation exposure andcancer mortality in the Techa River Cohort. Radiat. Res. 164, 602-611.
Kreuzer, M., Kreienbrock, L., Muller, K.M., et al., 1999. Histologic types of lung carcinoma and age
at onset. Cancer 85, 1958-1965.
Krewski, D., Lubin, J.H., Zielinski, J.M., et al., 2005. Residential radon and risk of lung cancer: a
combined analysis of 7 North American case-control studies. Epidemiology 16, 137-145
Krishnan, V., Bryant, H.U., Macdougald, O.A., 2006. Regulation of bone mass by Wnt signaling. J.
Clin. Invest. 116, 1202-1209.
Krivtsov, A.V., Twomey, D., Feng, Z., et al., 2006. Transformation from committed progenitor toleukaemia stem cell initiated by MLL-AF9. Nature 442, 818-822.
Krohn, K., Maier, J., Paschke, R., 2007. Mechanisms of disease: hydrogen peroxide, DNA damage
and mutagenesis in the development of thyroid tumors. Endocrinol. Metab. 3, 713-720.
Kronenwett, R., Butterweck, U., Steidl, U., et al., 2005. Distinct molecular phenotype of malignant
CD34(+) hematopoietic stem and progenitor cells in chronic myelogenous leukemia. Oncogene
24, 5313-5324.
Kubota, K., Suda, T., 2008. Bone marrow long label-retaining cells reside in the sinusodial hypoxic
niche. Biochem. Biophys. Res. Commun. 366, 335-339.
Kühl, M., Sheldahl, L.C., Park, M., et al., 2000. The Wnt/Ca2+ pathway: a new vertegbrate Wnt
Signaling pathway takes shape. Trends Genet. 16, 279-283.
Kumagai, A., Namba, H., Saenko, V.A., et al., 2004. Low frequency of BRAFT1796A mutations inchildhood thyroid carcinomas. J. Clin. Endocrinol. Metab. 89, 4280-4284.
Kurpinski, K., Jang, D.J., Bhattacharya, S., et al., 2009. Differential effects of x-rays and high-energy(56)Fe ions on human mesenchymal stem cells. Int. J. Radiat. Oncol. Biol. Phys. 73, 869-877.
Kusakabe, T., Hoshi, N., Kimura, S., 2006. Origin of the ultimobranchial body cyst: T/ebp/Nkx2.1
expression is required for development and fusion of the ultimobranchial body to the thyroid.
Dev. Dyn. 235, 1300-1309.
Kusunoki, Y., Kodama, Y., Hirai, Y., et al., 1995. Cytogenetic and immunologic identification of
clonal expansion of stem cells into T and B lymphocytes in one Atomic-bomb survivor. Blood86, 2106-2112.
Kyoizumi, S., Akiyama, M., Cologne, J.B., et al., 1996. Somatic cell mutations at the glycophorin A
locus in erythrocytes of atomic bomb survivors: implications for radiation carcinogenesis.
Radiat. Res. 146, 43-52.
Kyoizumi, S., Kusunoki, Y., Hayashi, T., et al., 2005. Individual variation of somatic gene mutability
in relation to cancer susceptibility: prospective study on erythrocyte glycophorin a genemutations of atomic bomb survivors. Cancer Res. 65, 5462-5469.

250

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10648	  10649	
  10650	
  10651	
  10652	  10653	  10654	  10655	  
10656	  10657	  10658	  10659	
  10660	
  10661	  
10662	  
10663	  
10664	  
10665	
  10666	  
10667	  
10668	  
10669	
  10670	  10671	  10672	  10673	  10674	  10675	  
10676	  10677	  10678	  10679	
  10680	  10681	  10682	  10683	  10684	  10685	
  10686	  10687	  10688	  10689	
  10690	  
10691	  
10692	  
10693	  
10694	  
10695	  
10696	  10697	  
10698	
  10699	
  10700	
  Lafuma, J., Chmelevsky, D., Chameaud, J., et al., 1989. Lung carcinomas in Sprague-Dawley rats
after exposure to low doses of radon daughters, fission neutrons, or gamma rays. Radiat. Res.
118, 230-245.
Laidlaw, I.J., Clarke, R.B., Howell, A., et al., 1995. The proliferation of normal human breast tissue
implanted into athymic nude mice is stimulated by estrogen but not progesterone.
Endocrinology 136, 164-171.
Lam, D.C., Girard, L., Suen, W.S., et al., 2006. Establishment and expression profiling of new lungcancer cell lines from Chinese smokers and lifetime never-smokers. J. Thorac. Oncol. 1, 932942.
Lan, L., Cui, D., Nowka, K., et al., 2007. Stem cells derived from goiters in adults form spheres in
response to intense growth stimulation and require thyrotropin for differentiation into
thyrocytes. J. Clin. Endocrinol. Metab. 92, 3681-3688.
Land, C.E., Hayakawa, N., Machado, S.G., et al., 1994. A case-control interview study of breast
cancer among Japanese A-bomb survivors. I. Main effects. Cancer Causes Control 5, 157-165.
Land, C.E., Shimosato, Y., Saccomanno, G., et al., 1993. Radiation-associated lung cancer: acomparison of the histology of lung cancers in uranium miners and survivors of the atomic
bombings of Hiroshima and Nagasaki. Radiat. Res. 134, 234-243.
Lane, S., Rippon, H.J., Bishop, A.E., 2007. Stem cells in lung repair and regeneration. Regen. Med. 2,407-415.
Lange, C., Brunswig-Spickenheier, B., Cappallo-Obermann, H., et al., 2011. Radiation rescue:mesenchymal stromal cells protect from lethal irradiation. PloS One 6, e14486.
Lansdorp, P.M., 2007. Immortal strands? Give me a break. Cell 129, 1244-1247.
Lantuejoul, S., Soria, J.C., Moro-Sibilot, D., et al., 2004. Differential expression of telomerase reverse
transcriptase (hTERT) in lung tumours. Br. J. Cancer 90, 1222-1229.
Lapidot, T., Sirard, C., Vormoor, J., et al., 1994. A cell initiating human acute myeloid leukaemia
after transplantation into SCID mice. Nature 367, 645-648.
Larcher, F., Dellambra, E., Rico, L., et al., 2007. Long-term engraftment of single geneticallymodified human epidermal holoclones enables safety pre-assessment of cutaneous gene therapy.
Mol. Ther. 15, 1670-1676.
Larderet, G., Fortunel, N.O., Vaigot, P., et al., 2006. Human side population keratinocytes exhibit
long-term proliferative potential and a specific gene expression profile and can form apluristratified epidermis. Stem Cells 24, 965-974.
Lawson, D.A., Witte, O.N., 2007. Stem cells in prostate cancer initiation and progression. J. Clin.
Invest. 117, 2044-2050.
Lawson, J.S., Field, A.S., Champion, S., et al., 1999. Low oestrogen receptor alpha expression innormal breast tissue underlies low breast cancer incidence in Japan. Lancet 354, 1787-1788.
Lawson, J.S., Field, A.S., Tran, D.D., et al., 2001. Hormone replacement therapy use dramaticallyincreases breast oestrogen receptor expression in obese postmenopausal women. Breast Cancer
Res. 3, 342-345.
Lawson, J.S., Field, A.S., Tran, D.D., et al., 2002. Breast cancer incidence and estrogen receptor alphain normal mammary tissue--an epidemiologic study among Japanese women in Japan and
Hawaii. Int. J. Cancer 97, 685-687.
Le Calvez, F., Mukeria, A., Hunt, J.D., et al., 2005. TP53 and Kras mutation load and types in lungcancers in relation to tobacco smoke: distinct patterns in never, former, and current smokers.
Cancer Res. 65, 5076-5083.
Leach, F.S., Tokino, T., Meltzer, P., et al., 1993. p53 Mutation and MDM2 amplification in humansoft tissue sarcomas. Cancer Res. 53, 2231-2234.
Lebedeva, G.A., 1973. [Intestinal polyps occurring under the influence of various types of ionizing
radiation]. Voprosy Onkologii 19, 47-51.
Lechler, T., Fuchs, E., 2005. Asymmetric cell divisions promote stratification and differentiation of
mammalian skin. Nature 437, 275-280.
Lee, H.W., Blasco, M.A., Gottlieb, G.J., et al., 1998. Essential role of mouse telomerase in highly
proliferative organs. Nature 392, 569-574.

251

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10701	  
10702	
  
10703	  
10704	  
10705	  
10706	  
10707	  
10708	  
10709	
  10710	  10711	
  10712	
  10713	  
10714	  
10715	  
10716	  
10717	  
10718	  
10719	
  10720	
  10721	  
10722	  
10723	  
10724	  
10725	  
10726	
  10727	  
10728	  
10729	
  10730	
  10731	
  
10732	
  
10733	  
10734	  
10735	  
10736	  10737	  
10738	  
10739	
  10740	  
10741	  
10742	  
10743	  10744	  10745	  
10746	  10747	  
10748	  
10749	
  10750	
  10751	  Lee, W., Chiacchierini, R.P., Shleien, B., et al., 1982. Thyroid tumors following 131I or localized X
irradiation to the thyroid and pituitary glands in rats. Radiat. Res. 92, 307-319.
Lee, W., Shleien, B., Telles, N.C., et al., 1979. An accurate method of 131I dosimetry in the rat thyroid.
Radiat. Res. 79, 55-62.
Leggett, R.W., Eckerman, K.F., Williams, L.R., 1982. Strontium-90 in bone: a case study in agedependent dosimetric modeling. Health Physics 43, 307-322.
Leng, S., Stidley, C.A., Willink, R., et al., 2008. Double-strand break damage and associated DNA
repair genes predispose smokers to gene methylation. Cancer Res. 68, 3049-3056.
Levi, F., Moeckli, R., Randimbison, L., et al., 2006a. Skin cancer in survivors of childhood andadolescent cancer. Eur. J. Cancer 42, 656-659.
Levi, F., Randimbison, L., Maspoli, M., et al., 2006b. High incidence of second basal cell skincancers. Int. J. Cancer 119, 1505-1507.
Levy, D.B., Smith, K.J., Beazer-Barclay, Y., et al., 1994. Inactivation of both APC alleles in humanand mouse tumors. Cancer Res. 54, 5953-5958.
Levy, O., Dai, G., Riedel, C., et al., 1997. Characterization of the thyroid Na+/I- symporter with ananti-COOH terminus antibody. Proc. Natl. Acad. Sci. U S A. 94, 5568-5573.
Li, A., Simmons, P.J., Kaur, P., 1998. Identification and isolation of candidate human keratinocyte
stem cells based on cell surface phenotype. Proc. Natl. Acad. Sci. U S A. 95, 3902-3907.
Li, J., Kwong, D.L., Chan, G.C., 2007. The effects of various irradiation doses on the growth anddifferentiation of marrow-derived human mesenchymal stromal cells. Pediatr. Transplant 11,
379-387.
Li, J., Miao, C., Guo, W., et al., 2008. Enrichment of putative human epidermal stem cells based oncell size and collagen type IV adhesiveness. Cell Res. 18, 360-371.
Li, T.S., Marban, E., 2010. Physiological levels of reactive oxygen species are required to maintaingenomic stability in stem cells. Stem Cells 28, 1178-1185.
Li, Y.Q., Fan, C.Y., O'Connor, P.J., et al., 1992. Target cells for the cytotoxic effects of carcinogens
in the murine small bowel. Carcinogenesis 13, 361-368.
Liang, X., So, Y.H., Cui, J., et al., 2011. The low-dose ionizing radiation stimulates cell proliferation
via activation of the MAPK/ERK pathway in rat cultured mesenchymal stem cells. J. Radiat.
Res. 52, 380-386.
Lichter, M.D., Karagas, M.R., Mott, L.A., et al., 2000. Therapeutic ionizing radiation and theincidence of basal cell carcinoma and squamous cell carcinoma. The New Hampshire Skin
Cancer Study Group. Arch. Dermatol. 136, 1007-1011.
Lidonnici, M.R., Corradini, F., Waldron, T., et al., 2008. Requirement of c-Myb for p210(BCR/ABL)dependent transformation of hematopoietic progenitors and leukemogenesis. Blood 111, 47714779.
Likhtarev, I.A., Sobolev, B.G., Kairo, I.A., et al., 1995. Thyroid cancer in the Ukraine. Nature 375,
365.
Lim, E., Wu, D., Pal, B., et al., 2010. Transcriptome analyses of mouse and human mammary cell
subpopulations reveal multiple conserved genes and pathways. Breast Cancer Res. 12, R21.
Lima, J., Trovisco, V., Soares, P., et al., 2004. BRAF mutationa are not a major event in postChernobyl childhood thyroid carcinomas. J. Clin. Endocrinol. Metab. 89, 4267-4271.
Lin, Q., Lee, Y.J., Yun, Z., 2006. Differentiation arrest by hypoxia. J. Biol. Chem. 281, 30678-30683.
Lin, R.Y., 2007. New insights in thyroid stem cells. Thyroid 17, 1019-1023.
Lin, R.Y., Kubo, A., Keller, G.M., et al., 2003. Committing embryonic stem cells to differentiate into
thyrocyte-like cells in vitro. Endocrinol. 144, 2644-2649.
Little, M.P., 2009. Cancer and non-cancer effects in Japanese atomic bomb survivors. J. Radiol. Prot.
29, A43-59.
Liu, B.Y., McDermott, S.P., Khwaja, S.S., et al., 2004. The transforming activity of Wnt effectors
correlates with their ability to induce the accumulation of mammary progenitor cells. Proc. Natl.
Acad. Sci. U S A. 101, 4158 - 4163.

252

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10752	  10753	  10754	  
10755	  
10756	  10757	  10758	  
10759	
  10760	
  10761	  
10762	  
10763	  
10764	  
10765	
  10766	  
10767	  
10768	  
10769	
  10770	  
10771	  
10772	  
10773	  
10774	  
10775	  
10776	  
10777	  
10778	  
10779	
  10780	  
10781	  
10782	  
10783	  
10784	  
10785	
  10786	  
10787	  
10788	  
10789	
  10790	  
10791	  
10792	  
10793	  
10794	  
10795	  
10796	  
10797	  
10798	
  10799	
  10800	
  10801	
  
10802	
  
10803	  Liu, C., Kawata, T., Shigematsu, N., et al., 2010. A comparison of chromosome repair kinetics inG(0) and G(1) reveals that enhanced repair fidelity under noncycling conditions accounts forincreased potentially lethal damage repair. Radiat. Res. 174, 566-573.
Liu, J., Finkel, T., 2006. Stem cell aging: what bleach can teach. Nature Medicine 12, 383-384.
Lloyd, R.D., Taylor, G.N., Miller, S.C., et al., 2001. Review of 239Pu and 226Ra effects in beagles.
Health Phys. 81, 691-697.
Lo Celso, C., Berta, M.A., Braun, K.M., et al., 2008. Characterization of bipotential epidermal
progenitors derived from human sebaceous gland: contrasting roles of c-Myc and beta-catenin.
Stem Cells 26, 1241-1252.
Lo Muzio, L., 2008. Nevoid basal cell carcinoma syndrome (Gorlin syndrome). Orphanet J. Rare
Diseases 3, 32.
Lo, Y.M., Darby, S., Noakes, L., et al., 1995. Screening for codon 249 p53 mutation in lung cancer
associated with domestic radon exposure. Lancet 345, 60.
Lobrich, M., Jeggo, P.A., 2005. Harmonising the response to DSBs: a new string in the ATM bow.
DNA repair 4, 749-759.
Loeffler, M., Birke, A., Winton, D., et al., 1993. Somatic mutation, monoclonality and stochastic
models of stem cell organization in the intestinal crypt. J. Theor. Biol. 160, 471-491.
Logan, C.Y., Nusse, R., 2004. The Wnt signaling pathway in development and disease. Annu. Rev.
Cell. Dev. Biol. 20, 781-810.
Lopez-Garcia, C., Klein, A.M., Simons, B.D., et al., 2010. Intestinal stem cell replacement follows apattern of neutral drift. Science 330, 822-825.
Lord, B.I., 1965. Hemopoietic changes in the rat during growth and during continuous gamma
irradiation of the adult animal. Br. J. Haematol. 11, 525-536.
Lord, B.I., Austin, A.L., Ellender, M., et al., 2001. Tumorigenic target cell regions in bone marrowstudied by localized dosimetry of 239Pu, 241Am and 233U in the mouse femur. Int. J. Radiat. Biol.
77, 665-678.
Lorenz, R., Deubel, W., Leuner, K., et al., 1994. Dose and dose-rate dependence of the frequency of
HPRT deficient T lymphocytes in the spleen of the 137Cs gamma-irradiated mouse. Int. J.
Radiat. Biol. 66, 319-326.
Lorimore, S.A., Chrystal, J.A., Robinson, J.I., et al., 2008. Chromosomal instability in unirradiatedhemaopoietic cells induced by macrophages exposed in vivo to ionizing radiation. Cancer Res.
68, 8122-8126.
Lowry, W.E., Blanpain, C., Nowak, J.A., et al., 2005. Defining the impact of beta-catenin/Tcf
transactivation on epithelial stem cells. Genes & Development 19, 1596-1611.
Lu, F., Wong, C.S., 2005. A clonogenic survival assay of neural stem cells in rat spinal cord after
exposure to ionizing radiation. Radiat. Res. 163, 63-71.
Lubin, J.H., Boice, J.D., Jr., Edling, C., et al., 1995. Radon-exposed underground miners and inverse
dose-rate (protraction enhancement) effects. Health Phys. 69, 494-500.
Lundell, M., Mattsson, A., Karlsson, P., et al., 1999. Breast cancer risk after radiotherapy in infancy: apooled analysis of two Swedish cohorts of 17,202 infants. Radiat. Res. 151, 626-632.
Lundgren, D.L., Haley, P.J., Hahn, F.F., et al., 1995. Pulmonary carcinogenicity of repeatedinhalation exposure of rats to aerosols of 239PuO2. Radiat. Res. 142, 39-53.
Luongo, C., Moser, A.R., Gledhill, S., et al., 1994. Loss of Apc+ in intestinal adenomas from Min
mice. Cancer Res. 54, 5947-5952.
Luz, A., Gossner, W., Heuck, 1991. Bone, Strahlengefahrdung und Strahlenschutz/Radiation
Exposure and Radiation Protection. Springer-Verlag, Heidelberg, pp. 265-316.
Lyle, S., Christofidou-Solomidou, M., Liu, Y., et al., 1998. The C8/144B monoclonal antibody
recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. J. Cell Sci.
111 ( Pt 21), 3179-3188.
Lynch, T.J., Bell, D.W., Sordella, R., et al., 2004. Activating mutations in the epidermal growth factor
receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med.
350, 2129-2139.

253

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10804	
  
10805	
  
10806	
  
10807	  
10808	  10809	
  10810	
  10811	
  10812	
  10813	
  10814	
  10815	  
10816	  10817	  10818	  10819	
  10820	
  10821	
  
10822	
  10823	
  
10824	
  10825	  
10826	
  10827	  10828	  10829	
  10830	
  10831	
  10832	
  10833	
  10834	
  10835	
  
10836	
  10837	  10838	  10839	
  10840	  10841	
  10842	
  10843	  10844	  10845	  
10846	  10847	  10848	  10849	
  10850	
  10851	
  10852	
  10853	
  10854	
  Lyon, C.M., Klinge, D.M., Liechty, K.C., et al., 2007. Radiation-induced lung adenocarcinoma is
associated with increased frequency of genes inactivated by promoter hypermethylation. Radiat.
Res. 168, 409-414.
M'Kacher, R., Laithier, V., Valent, A., et al., 2003. Sensitivity to radiation and alkylating agent of
peripheral lymphocytes and fibroblasts in a Hoyeraal-Hreidarsson syndrome patient. Pediatr.
Hematol. Oncol. 20, 651-656.
Ma, J., Shi, M., Li, J., et al., 2007. Senescence-unrelated impediment of osteogenesis from Flk1+
bone marrow mesenchymal stem cells induced by total body irradiation and its contribution to
long-term bone and hematopoietic injury. Haematologica 92, 889-896.
MacDonald, D., Boulton, E., Pocock, D., et al., 2001. Evidence of genetic instability in 3 Gy X-rayinduced mouse leukaemias and 3 Gy X-irradiated haemopoietic stem cells. Int. J. Radiat. Biol.
77, 1023-1031.
MacKenzie, I.C., 1975. Ordered structure of the epidermis. J. Invest. Dermatol. 65, 45-51.
MacKenzie, I.C., Bickenbach, J.R., 1985. Label-retaining keratinocytes and Langerhans cells inmouse epithelia. Cell Tissue Res. 242, 551-556.
MacKenzie, I.C., Zimmerman, K., Peterson, L., 1981. The pattern of cellular organization of humanepidermis. J. Invest. Dermatol. 76, 459-461.
Mackie, E.J., 2003. Osteoblasts: novel roles in orchestration of skeletal architecture. Int. J. Biochem.
Cell. Biol. 35, 1301-1305.
Maeda, H., 1998. Carcinogenesis via microbial infection. Gan To Kagaku Ryoho 25, 1474-1485.
Maes, J., Maleszewska, M., Guillemin, C., et al., 2008. Lymphoid-affiliated genes are associated withactive histone modifications in human hematopoietic stem cells. Blood 112, 2722-2729. Epub
2008 Jul 2714.
Maier, J., van Steeg, H., van Oostrom, C., et al., 2006. Deoxyribonucleic acid damage andspontaneous mutagenesis in the thyroid gland of rats and mice. Endocrinol. 147, 3391-3397.
Major, I.R., Mole, R.H., 1978. Myeloid leukaemia in x-ray irradiated CBA mice. Nature 272, 455-456.
Makino, S., Fukuda, K., Miyoshi, S., et al., 1999. Cardiomyocytes can be generated from marrowstromal cells in vitro. J. Clin. Invest. 103, 697-705.
Malanchi, I., Peinado, H., Kassen, D., et al., 2008. Cutaneous cancer stem cell maintenance isdependent on beta-catenin signalling. Nature 452, 650-653.
Malashicheva, A.B., Kislyakova, T.V., Aksenov, N.D., et al., 2000. F9 embryonal carcinoma cells failto stop at G1/S boundary of cell cycle after gamma-irradiation due to p21WAF1/CIP1
degradation. Oncogene 19, 3858-3865.
Mancuso, M., Gallo, D., Saran, A., 2009. Re: Modulation of basal and squamous cell carcinoma byendogenous estrogen in mouse models of skin cancer. Carcinogenesis 30, 721.
Mancuso, M., Pasquali, E., Leonardi, S., et al., 2008. Oncogenic bystander radiation effects in
Patched heterozygous mouse cerebellum. Proc. Natl. Acad. Sci. U S A. 105, 12445-12450.
Mancuso, M., Pazzaglia, S., Tanori, M., et al., 2004. Basal cell carcinoma and its development:
insights from radiation-induced tumors in Ptch1-deficient mice. Cancer Res. 64, 934-941.
Mangalik, A., Robinson, W.A., Morton, N.J., 1979. Stem cell kinetics in humans: studies during
autologous nonfrozen bone marrow transplantation. Exp. Hematol. 7, 188-199.
Mani, S.A., Guo, W., Liao, M.-J., et al., 2008. The epithelial-mesenchymal transition generates cellswith properties of stem cells. Cell 133, 704-715.
Manley, N.R., Capecchi, M.R., 1995. The role of Hoxa-3 in mouse thymus and thyroid development.
Development 121, 1989-2003.
Manolagas, S.C., 2000. Birth and death of bone cells: basic regulatory mechanisms and implications
for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 21, 115-137.
Mansouri, A., Chowdhury, K., Gruss, P., 1998. Follicular cells of the thyroid gland require Pax8 genefunction. Nat. Genet. 19, 87-90.
Mao, J.H., Li, J., Jiang, T., et al., 2005. Genomic instability in radiation-induced mouse lymphoma
from p53 heterozygous mice. Oncogene 24, 7924-7934.

254

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10855	  
10856	
  10857	  10858	  10859	
  10860	
  10861	  
10862	  
10863	  
10864	  
10865	
  10866	  
10867	  
10868	  
10869	
  10870	  10871	
  10872	
  10873	  10874	  10875	  
10876	  10877	  10878	  10879	
  10880	  
10881	  
10882	  
10883	  
10884	  
10885	
  10886	  
10887	  
10888	  
10889	
  10890	  
10891	  
10892	  
10893	  
10894	  
10895	  
10896	  
10897	  
10898	
  10899	
  10900	
  10901	
  10902	
  10903	
  10904	
  10905	
  
10906	
  Marchetti, A., Pellegrini, S., Sozzi, G., et al., 1998. Genetic analysis of lung tumours of non-smoking
subjects: p53 gene mutations are constantly associated with loss of heterozygosity at the FHIT
locus. Br. J. Cancer. 78, 73-78.
Marotta, V., Guerra, A., Sapio, M.R., et al., 2011. RET/PTC rearrangement in benign and malignant
thyroid diseases: a clinical standpoint. Europ. J. Endocrinol. 165, 499-507.
Marshman, E., Booth, C., Potten, C.S., 2002. The intestinal epithelial stem cell. Bioessays 24, 91-98.
Martin, G.R., 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium
conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U S A. 78, 7634-7638.
Martin, K., Kirkwood, T.B., Potten, C.S., 1998a. Age changes in stem cells of murine small intestinal
crypts. Exp. Cell Res. 241, 316-323.
Martin, K., Potten, C.S., Roberts, S.A., et al., 1998b. Altered stem cell regeneration in irradiatedintestinal crypts of senescent mice. J. Cell Sci. 111 ( Pt 16), 2297-2303.
Martins-Neves, S.R., Lopes, A.O., do Carmo, A., et al., 2012. Therapeutic implications of an enrichedcancer stem-like cell population in a human osteosarcoma cell line. BMC cancer 12, 139.
Marusyk, A., 2009. Irradiation alters selection for oncogenic mutations in hematopoietic progenitors.
Cancer Res., 7262-7269.
Marusyk, A., Porter, C.C., Zaberezhnyy, V., et al., 2010. Irradiation selects for p53-deficient
hematopoietic progenitors. PLoS Biol. 8.
Mason, K.A., Tofilon, P.J., 1994. Unexpect radiation protection with 13-cis-retinoic acid plus
interferon alpha-2a. Cancer Chemother. Pharmacol. 33, 435-437.
Masse, R., 1995. RBE for carcinogenesis following exposure to high LET radiation. Radiation andEnviron. Biophys. 34, 223-227.
Matakidou, A., Eisen, T., Houlston, R.S., 2005. Systematic review of the relationship between family
history and lung cancer risk. Br. J. Cancer 93, 825-833.
Mathon, N.F., Lloyd, A.C., 2001. Cell senescence and cancer. Nat. Rev. Cancer 1, 203-213.
Matsumoto, S., Iwakawa, R., Takahashi, K., et al., 2007. Prevalence and specificity of LKB1 genetic
alterations in lung cancers. Oncogene 26, 5911-5918.
Matthews, J.L., 1980. Bone structure and ultrastructure, Fundamental and clinical bone physiology.
J.B. Lippincott Co., Philadelphia, USA.
Mattison, R.J., Ostler, K.R., Locke, F.L., et al., 2007. Implications of FLT3 mutations in the therapyof acute myeloid leukemia. Rev. Recent Clin. Trials 2, 135-141.
Mattsson, A., Ruden, B.-I., Wilking, N., et al., 1993. Radiation-induced breast cancer: Long-term
follow-up of radiation therapy for benign breast disease. J. Natl. Cancer Inst. 85, 1679-1685.
Mattsson, J., Jansson, M., Wernerson, A., et al., 2004. Lung epithelial cells and type II pneumocytesof donor origin after allogeneic hematopoietic stem cell transplantation. Transplantation 78,
154-157.
Maxon, H.R., Saenger, E.L., 2000. Biological effects of radioiodines on the human thyroid gland.
Werner and Ingbar’s The Thyroid. Lippincott Williams and Wilkins, Philadelphia.
Maxon, H.R., Thomas, S.R., Saenger, E.L., et al., 1977. Ionizing irradiation and the induction ofclinically significant disease in the human thyroid gland. Am. J. Med. 63, 967-978.
May, R., Riehl, T.E., Hunt, C., et al., 2008. Identification of a novel putative gastrointestinal stem celland adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury
and in adenomatous polyposis coli/multiple intestinal neoplasia mice. Stem cells 26, 630-637.
McBeath, R., Pirone, D.M., Nelson, C.M., et al., 2004. Cell shape, cytoskeletal tension and RhoA
regulate stem cell lineage commitment. Am. J. Physiol. 275, 1898-1908.
McCord, A.M., Jamal, M., Williams, E.S., et al., 2009. CD133+ glioblastoma stem-like cells areradiosensitive with a defective DNA damage response compared with established cell lines.
Clin. Cancer Res. 15, 5145-5153.
McCroskery, S., Thomas, M., Maxwell, L., et al., 2003. Myostatin negatively regulates satellite cell
activation and self-renewal. J. Cell. Biol. 162, 1135-1147.
McIlrath, J., Lorimore, S.A., Coates, P.J., et al., 2003. Radiation-induced genomic instability inimmortalized haemopoietic stem cells. Int. J. Radiat. Biol. 79, 27-34.

255

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10907	
  10908	  10909	
  10910	
  10911	
  10912	
  10913	  10914	  10915	  10916	  10917	  10918	  10919	
  10920	
  10921	
  10922	
  10923	  10924	  10925	  10926	
  10927	  10928	  10929	
  10930	
  10931	
  10932	
  10933	  10934	  10935	  10936	  10937	  10938	  10939	
  10940	  10941	
  10942	  10943	  10944	  10945	  10946	  10947	  10948	  10949	
  10950	
  10951	
  10952	  10953	  10954	  10955	  10956	  10957	  10958	  Medina, D., Kittrell, F.S., Shepard, A., et al., 2002. Biological and genetic properties of the p53 nullpreneoplastic mammary epithelium. FASEB J 16, 881-883.
Medina, D., Oborn, C.J., Kittrell, F.S., et al., 1986. Properties of mouse mammary epithelial cell linescharacterized by in vivo transplantation and in vitro immunocytochemical methods. J. Natl.
Cancer. Inst. 76, 1143-1156.
Mehrara, B.J., Avraham, T., Soares, M., et al., 2010. p21cip/WAF is a key regulator of long-term
radiation damage in mesenchyme-derived tissues. FASEB J 24, 4877-4888.
Meijne, E.I., Ploemacher, R.E., Huiskamp, R., 1996. Sensitivity of murine haemopoietic stem cell
populations to X-rays and 1 MeV fission neutrons in vitro and in vivo under hypoxic conditions.
Int. J. Radiat. Biol. 70, 571-577.
Meijne, E.I., van der Winden-van Groenewegen, R.J., Ploemacher, R.E., et al., 1991. The effects of xirradiation on hematopoietic stem cell compartments in the mouse. Exp. Hematol. 19, 617-623.
Meissner, A., Wernig, M., Jaenisch, R., 2007. Direct reprogramming of genetically unmodified
fibroblasts into pluripotent stem cells. Nat. Biotechnol. 25, 1177-1181.
Mercer, R.R., Russell, M.L., Roggli, V.L., et al., 1994. Cell number and distribution in human and rat
airways. Am. J. Respir. Cell. Mol. Biol. 10, 613-624.
Merritt, A.J., Potten, C.S., Watson, A.J., et al., 1995. Differential expression of bcl-2 in intestinal
epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence ofcolonic neoplasia. J. Cell Sci. 108 (Pt 6), 2261-2271.
Mertens, A.C., Liu, Q., Neglia, J.P., et al., 2008. Cause-specific late mortality among 5-year survivorsof childhood cancer: the childhood cancer survivor study. J. Nat. Canc. Inst. 100, 1368-1379.
Metcalf, D., 1977. Hematopoietic cells. Springer-Verlag, Berlin.
Metcalf, D., 2003. The unsolved enigmas of leukemia inhibitory factor. Stem Cells 21, 5-14.
Metcalf, D., Moore, M., 1971. Hemopoietic cells. Frontiers of Biology 24.
Mettler, F.A., Upton, A.C., 1995. Medical effects of ionizing radiation. 2nd edition. W.B. SaundersCo., Philadelphia.
Midgley, C.A., Owens, B., Briscoe, C.V., et al., 1995. Coupling between gamma irradiation, p53
induction and the apoptotic response depends upon cell type in vivo. J. Cell Sci. 108 ( Pt 5),
1843-1848.
Miller, C.L., Eaves, C.J., 1997. Expansion in vitro of adult murine hematopoietic stem cells withtransplantable lympho-myeloid reconstituting ability. Proc. Natl. Acad. Sci. U S A. 94, 1364813653.
Miller, R.C., Hiraoka, T., Kopecky, K.J., et al., 1987. Sensitivity to radiation of human normal,
hyperthyroid, and neoplastic thyroid epithelial cells in primary culture. Radiat. Res. 111, 81-91.
Minna, J.D., Roth, J.A., Gazdar, A.F., 2002. Focus on lung cancer. Cancer Cell 1, 49-52.
Minucci, S., Monestiroli, S., Giavara, S., et al., 2002. PML-RAR induces promyelocytic leukemiaswith high efficiency following retroviral gene transfer into purified murine hematopoietic
progenitors. Blood 100, 2989-2995.
Mitchell, G., Farndon, P.A., Brayden, P., et al., 2005. Genetic predisposition to cancer: theconsequences of a delayed diagnosis of Gorlin syndrome. Clin. Oncol. (R. Coll. Radiol.) 17,
650-654.
Miyamoto, T., Nagafuji, K., Akashi, K., et al., 1996. Persistence of multipotent progenitors expressing
AML1/ETO transcripts in long-term remission patients with t(8;21) acute myelogenous
leukemia. Blood 87, 4789-4796.
Miyoshi-Imamura, T., Kakinuma, S., Kaminishi, M., et al., 2010. Unique characteristics of radiationinduced apoptosis in the postnatally developing small intestine and colon of mice. Radiat. Res.
173, 310-318.
Mizuno, T., Iwamoto, K.S., Kyoizumi, S., et al., 2000. Preferential induction of RET/PTC
rearrangement by x-ray irradiation. Oncogene 19, 438-443.
Mizuno, T., Tokuoka, S., Kishikawa, M., et al., 2006. Molecular basis of basal cell carcinogenesis in
the atomic-bomb survivor population: p53 and PTCH gene alterations. Carcinogenesis 27,2286-2294.

256

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
10959	
  10960	
  10961	  
10962	  10963	  10964	  10965	
  10966	  10967	  
10968	  
10969	
  10970	  10971	  10972	  10973	  10974	  10975	  10976	  10977	  10978	  10979	
  10980	  10981	  10982	  10983	  10984	  10985	
  10986	  10987	  
10988	  10989	
  10990	  
10991	  
10992	  
10993	  
10994	  
10995	  
10996	  10997	  
10998	
  10999	
  11000	
  11001	
  11002	
  11003	
  11004	
  11005	
  11006	
  11007	
  11008	
  11009	
  11010	
  11011	
  Modlin, I.M., Kidd, M., Lye, K.D., et al., 2003. Gastric stem cells: an update. The Keio Journal of
Medicine 52, 134-137.
Mohrin, M., Bourke, E., Alexander, D., et al., 2010. Hematopoietic stem cell quiescence promotes
error-prone DNA repair and mutagenesis. Cell Stem Cell 7, 174-185.
Mole, R.H., 1990. Childhood cancer after prenatal exposure to diagnostic X-ray examinations in
Britain. Br. J. Cancer 62, 152-168.
Mole, R.H., Major, I.R., 1983. Myeloid leukaemia frequency after protracted exposure to ionizing
radiation: experimental confirmation of the flat dose-response found in ankylosing spondylitis
after a single treatment course with X-rays. Leukemia Res. 7, 295-300.
Mole, R.H., Papworth, D.G., Corp, M.J., 1983. The dose-response for x-ray induction of myeloid
leukaemia in male CBA/H mice. Br. J. Cancer 47, 285-291.
Molineux, G., Testa, N.G., Hendry, J.H., et al., 1987. The radiation sensitivity of the haemopoietic
microenvironment--effect of dose rate on ectopic ossicle formation. Radiother. and Oncol. 10,
157-161.
Montgomery, R.K., Carlone, D.L., Richmond, C.A., et al., 2011. Mouse telomerase reverse
transcriptase (mTert) expression marks slowly cycling intestinal stem cells. Proc. Natl. Acad.
Sci. U S A. 108, 179-184.
Moolgavkar, S.H., Cross, F.T., Luebeck, G., et al., 1990. A two-mutation model for radon-induced
lung tumors in rats. Radiat. Res. 121, 28-37.
Moon, R.T., Bowerman, B., Boutros, M., et al., 2002. The promise and perils of Wnt signaling
through beta-catenin. Science 296, 1644-1646.
Morgan, A., Black, A., 1989. Lung retention and translocation of Pu in mice following inhalation of
PuO2 and PuO2 fired at 550-1250 degrees. Radiation Prot. Dosim. 26, 297-301.
Morgan, J.E., Gross, J.G., Pagel, C.N., et al., 2002. Myogenic cell proliferation and generation of areversible tumorigenic phenotype are triggered by preirradiation of the recipient site. J. Cell.
Biol. 157, 693-702.
Morgan, W.F., Bair, W.J., 2013. Issues in low dose radiation biology: the controversy continues. A
perspective. Radiat. Res.179, 501-510.
Mori, H., Colman, S.M., Xiao, Z., et al., 2002. Chromosome translocations and covert leukemic
clones are generated during normal fetal development. Proc. Natl. Acad. Sci. U S A. 99, 82428247.
Moriconi, F., Malik, I., Ahmad, G., et al., 2009. Effect of irradiation on gene expression of rat liveradhesion molecules: in vivo and in vitro studies. Strahlenther. Onkol. 185, 460-468.
Morikawa, S., Mabuchi, Y., Kubota, Y., et al., 2009. Prospective identification, isolation, andsystemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J. Exp.
Med. 206, 2483-2496.
Morin Doody, M., Lonstein, J.E., Stovall, M., et al., 2000. Breast cancer mortality after diagnostic
radiography: findings from the U.S. Scoliosis Cohort Study. Spine 25, 2052-2063.
Morris, R.J., Fischer, S.M., Slaga, T.J., 1985. Evidence that the centrally and peripherally locatedcells in the murine epidermal proliferative unit are two distinct cell populations. J. Invest.
Dermatol. 84, 277-281.
Morris, R.J., Tryson, K.A., Wu, K.Q., 2000. Evidence that the epidermal targets of carcinogen action
are found in the interfollicular epidermis of infundibulum as well as in the hair follicles. Cancer
Res. 60, 226-229.
Moss, 1966. Bone. In: Greep, R.O. (Ed.), Histology 2nd Edition. McGraw-Hill, New York, pp. 155173.
Mouiseddine, M., Francois, S., Semont, A., et al., 2007. Human mesenchymal stem cells home
specifically to radiation-injured tissues in a non-obese diabetes/severe combined
immunodeficiency mouse model. Br. J. Radiol. 80, Spec No 1, S49-55.
Muggenburg, B.A., Hahn, F.F., Griffith, W.C., et al., 1995. The biological effects of 224Ra injected
into dogs. In: van Kaick, G., Karaoglou, A., Kellerer, A.M. (Eds.), Health Effects of InternallyDeposited Radionuclides: Emphasis on Radium and Thorium. World Scientific, Singapore, pp.
299-305.

257

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11012	
  11013	
  11014	  11015	
  11016	  11017	
  11018	  11019	
  11020	
  11021	
  11022	
  11023	
  11024	
  11025	
  
11026	
  11027	  11028	  11029	
  11030	
  11031	
  11032	
  11033	
  11034	
  11035	
  
11036	
  11037	
  11038	  11039	
  11040	
  11041	
  11042	
  11043	
  11044	
  11045	
  
11046	  11047	  11048	  11049	
  11050	
  11051	
  11052	
  11053	
  11054	
  11055	
  11056	  11057	  11058	  11059	
  11060	
  11061	
  
11062	
  
11063	  Muirhead, C., Cox, R., Stather, J., et al., 1993. Estimates of late radiation risks to the UK population.
Documents of the NRPB 4, 15-157.
Mulcahy, R.T., Gould, M.N., Clifton, K.H., 1980. The survival of thyroid cells: in vivo irradiation andin situ repair. Radiat. Res. 84, 523-528.
Mullenders, L., Atkinson, M., Paretzke, H., et al., 2009. Assessing cancer risks of low-dose radiation.
Nat. Rev. Cancer. 9, 596-604.
Muller, W.A., Luz, A., Murray, A.B., et al., 1990. Induction of lymphoma and osteosarcoma in miceby single and protracted low alpha doses. Health Physics 59, 305-310.
Murdoch, B., Chadwick, K., Martin, M., et al., 2003. Wnt-5A augments repopulating capacity andprimitive hematopoietic development of human blood stem cells in vivo. Proc. Natl. Acad. Sci.
U S A. 100, 3422-3427.
Murnane, J.P., 2012. Telomere dysfunction and chromosome instability. Mutat Res. 730, 28-36.
Muscat, J.E., Wynder, E.L., 1995. Lung cancer pathology in smokers, ex-smokers and never smokers.
Cancer Lett 88, 1-5.
Mussano, F., Lee, K.J., Zuk, P., et al., 2010. Differential effect of ionizing radiation exposure on
multipotent and differentiation-restricted bone marrow mesenchymal stem cells. J. Cell
Biochem. 111, 322-332.
Mutoh, H., Fung, B.P., Naya, F.J., et al., 1997. The basic helix-loop-helix transcription factor
BETA2/NeuroD is expressed in mammalian enteroendocrine cells and activates secretin gene
expression. Proc. Natl. Acad. Sci. U S A. 94, 3560-3564.
Nakabeppu, Y., Sakumi, K., Sakamoto, K., et al., 2006. Mutagenesis and carcinogenesis caused by
the oxidation of nucleic acids. Biol. Chem. 387, 373-379.
Nakajima, M., Kawanami, O., Jin, E., et al., 1998. Immunohistochemical and ultrastructural studies ofbasal cells, Clara cells and bronchiolar cuboidal cells in normal human airways. Pathol. Int. 48,
944-953.
Nakamura, N., 2005. A hypothesis: radiation-related leukemia is mainly attributable to the small
number of people who carry pre-existing clonally expanded preleukemic cells. Radiat. Res. 163,258-265.
Nakamura, Y., 1997. Cleaning up on beta-catenin. Nature Med. 3, 499-500.
Nakano, M., Kodama, Y., Ohtaki, K., et al., 2012. Translocations in spleen cells from adult mice
irradiated as fetuses are infrequent, but often clonal in nature. Radiat. Res. 178, 600-603.
Nakano, M., Kodama, Y., Ohtaki, K., et al., 2007. Chromosome aberrations do not persist in thelymphocytes or bone marrow cells of mice irradiated in utero or soon after birth. Radiat. Res.
167, 693-702.
Nakashima, T., Hayashi, M., Fukunaga, T., et al., 2011. Evidence for osteocyte regulation of bonehomeostasis through RANKL expression. Nat. Med. 17, 1231-1234.
Nakatsuka, H., Shimizu, Y., Yamamoto, T., et al., 1992. Colorectal cancer incidence among atomic
bomb survivors, 1950-80. J. Radiat. Res. 33, 342-361.
Nakeff, A., McLellan, W.L., Bryan, J., et al., 1979. Response of megakaryocyte, erythroid, andgranulocyte-macrophage progenitor cells in mouse bone marrow to gamma-irradiation andcyclophosphamide. Exp. Hematol., 99-110.
Namba, H., Hara, T., Tukazaki, T., et al., 1995. Radiation-induced G1 arrest is selectively mediatedby the p53-WAF1/Cip1 pathway in human thyroid cells. Cancer Res. 55, 2075-2080.
Nana-Sinkam, S.P., Geraci, M.W., 2006. MicroRNA in lung cancer. J. Thorac. Oncol. 1, 929-931.
Naruke, Y., Nakashima, M., Suzuki, K., et al., 2009. Genomic instability in the epidermis induced byatomic bomb radiation: a long-lasting health effect in atomic-bomb survivors. Cancer 115,
3782-3790.
NCRP-64, 1980. Influence of dose and its distribution in time on dose-response relationships for lowLET radiations. National Council on Radiation Protection and Measurements, Bethesda, MD.
NCRP-150, 2005. Extrapolation of radiation-induced cancer risks from nonhuman experimental
systems to humans, NCRP Report 150. National Council on Radiation Protection andMeasurements, Bethesda, MD, pp. 38-56.

258

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11064	  
11065	
  11066	  
11067	  
11068	  
11069	
  11070	  11071	  11072	
  11073	  11074	  11075	  
11076	  11077	  11078	  11079	
  11080	  11081	  
11082	
  
11083	  
11084	  
11085	
  11086	  
11087	  
11088	  
11089	
  11090	  
11091	
  
11092	
  
11093	  
11094	  
11095	  
11096	  
11097	  
11098	
  11099	
  11100	
  11101	
  11102	
  11103	
  11104	
  11105	
  
11106	
  11107	
  11108	
  11109	
  11110	
  11111	
  11112	
  11113	
  11114	
  11115	
  11116	  NCRP-174, 2014. Preconception and prenatal radiation exposure: health effects and protective
guidance (2013). National Council on Radiation Protection and Measurements, Bethesda, MD,
USA.
Nekolla, E.A., Kellerer, A.M., Kuse-Isingschulte, M., et al., 1999. Malignancies in patients treatedwith high doses of radium-224. Radiat. Res. 152, S3-7.
Nekolla, E.A., Kreisheimer, M., Kellerer, A.M., et al., 2000. Induction of malignant bone tumors inradium-224 patients: risk estimates based on the improved dosimetry. Radiat. Res. 153, 93-103.
Nekolla, E.A., Walsh, L., Schottenhammer, G., et al., 2005. Malignancies in patients treated with highdose of Ra-224. GSF- Forschungszentrum, Neuherberg Germany.
Neuman, W.F., 1980. Bone material and calcification mechanisms. Fundamental and clinical bone
physiology. J.B. Lippincott Co., Philadelphia, pp. 83-107.
Neuringer, I.P., Randell, S.H., 2004. Stem cells and repair of lung injuries. Respir. Res. 5, 6.
Neville, M.C., Daniel, C.W., 1987. The Mammary Gland: Development, Regulation and Function.
Plenum Press Publishing Corp, New York.
Nguyen, D.H., Fredlund, E., Zhao, W., et al., 2013. Murine microenvironment metaprofiles associatewith human cancer etiology and intrinsic subtypes. Clin. Cancer Res. 19, 1353-1362.
Nguyen, D.H., Oketch-Rabah, H.A., Illa-Bochaca, I., et al., 2011. Radiation acts on the
microenvironment to affect breast carcinogenesis by distinct mechanisms that decrease cancer
latency and affect tumor type. Cancer Cell 19, 640-651.
Nicholson, R.I., Gee, J.M., Harper, M.E., 2001. EGFR and cancer prognosis. Eur. J. Cancer 37 Suppl
4, S9-15.
NIH Report, 2008. Stem Cell Information, Chapter 5 in Hematopoietic stem cells.
cells.nih.gov/info/scireport/chapter5.asp. 8-4-2008.
Nijnik, 2007. DNA repair is limiting for haematopoietic stem cells during ageing. Nature 447, 686690.
Nikiforov, Y.E., Rowland, J.M., Bove, K.E., et al., 1997. Distinct pattern of ret oncogene
rearrangements in morphological variants of radiation induced and sporadic thyroid papillarycarcinomas in children. Cancer Res. 57, 1690-1694.
Nikiforova, M.N., Ciampi, R., Salvatore, G., et al., 2004. Low prevalence of BRAF mutations inradiation-induced thyroid tumors in contrast to sporadic papillary carcinomas. Cancer Lett 209,
1-6.
Nikiforova, M.N., Stringer, J.R., Blough, R., et al., 2000. Proximity of chromosomal loci thatparticipate in radiation induced rearrangements in human cells. Science 290, 138-141.
Nishikawa, S., Osawa, M., 2007. Generating quiescent stem cells. Pigment Cell Res. 20, 263-270.
Nomura, T., Hongyo, T., Nakajima, H., et al., 2008. Differential radiation sensitivity to morphological,
functional and molecular changes of human thyroid tissues and bone marrow maintained in
SCID mice. Mut. Res. 657, 68-76.
Notta, F., Doulatov, S., Laurenti, E., et al., 2011. Isolation of single human hematopoietic stem cells
capable of long-term multilineage engraftment. Science 333, 218-221.
Nouspikel, T., Hanawalt, P.C., 2002. DNA repair in terminally differentiated cells. DNA repair 1, 5975.
Nuciforo, P.G., Luise, C., Capra, M., et al., 2007. Complex engagement of DNA damage response
pathways in human cancer and in lung tumor progression. Carcinogenesis 28, 2082-2088.
O'Malley, S., Weitman, D., Olding, M., et al., 1997. Multiple neoplasms following craniospinal
irradiation for medulloblastoma in a patient with nevoid basal cell carcinoma syndrome. Case
report. J. Neurosurg. 86, 286-288.
Oghiso, Y., Yamada, Y., Ishigure, N., et al., 1994. High incidence of malignant lung carcinomas inrats after inhalation of 239PuO2 aerosol. J. Radiat. Res. 35, 222-235.
Ohtaki, K., Kodama, Y., Nakano, M., et al., 2004. Human fetuses do not register chromosome damage
inflicted by radiation exposure in lymphoid precursor cells except for a small but significant
effect at low doses. Radiat. Res. 161, 373-379.
Ohyama, M., Terunuma, A., Tock, C.L., et al., 2006. Characterization and isolation of stem cellenriched human hair follicle bulge cells. J. Clin. Invest. 116, 249-260.

259

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11117	
  11118	  11119	
  11120	
  11121	
  11122	
  11123	
  11124	
  11125	
  11126	
  11127	
  11128	
  11129	
  11130	
  11131	
  11132	
  11133	
  11134	
  11135	
  11136	
  11137	
  11138	
  11139	
  11140	
  11141	
  11142	
  11143	
  11144	
  11145	
  11146	
  11147	  11148	  11149	
  11150	
  11151	
  11152	
  11153	
  11154	
  11155	
  11156	
  11157	  11158	
  11159	
  11160	
  11161	
  11162	
  11163	  11164	
  11165	
  11166	
  11167	  11168	  
11169	  Okumoto, M., Nishikawa, R., Iwai, M., et al., 1990. Lack of evidence for the involvement of type-C
and type-B retroviruses in radiation leukemogenesis of NFS mice. Radiat. Res. 121, 267-273.
Oliai C., Yang L.X., 2013. Radioprotectants to reduce the risk of radiation-induced carcinogenesis. Int.
J. Radiat. Biol. Early online publication, November 2013.
Olivier, M., Petitjean, A., Marcel, V., et al., 2009. Recent advances in p53 research: aninterdisciplinary perspective. Cancer Gene Ther. 16, 1-12.
Ootani, A., Li, X., Sangiorgi, E., et al., 2009. Sustained in vitro intestinal epithelial culture within a
Wnt-dependent stem cell niche. Nature Med. 15, 701-706.
Opferman, J.T., Iwasaki, H., Ong, C.C., et al., 2005. Obligate role of anti-apoptotic MCL-1 in thesurvival of hematopoietic stem cells. Science 307, 1101-1104.
Ordonez, N.G., 2000. Thyroid transcription factor-1 is a marker of lung and thyroid carcinomas. Adv.
Anat. Pathol. 7, 123-127.
Orkin, S.H., Zon, L.I., 2008. Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132,631-644.
Otsu, H., Furuse, T., Noda, Y., et al., 1995. Leukemogenesis in C3H mice following continuous
irradiation with gamma rays or high and low dose rate. ICRR '95 Congress Proceedings:
Radiation Research 1895-1995. Universitatsdruckerei H Stutz AG, Wurzburg, Germany, pp.
743-748.
Ottaviani, G., Jaffe, N., 2009. The epidemiology of osteosarcoma. Cancer Treat Res. 152, 3-13.
Owens, D.M., Watt, F.M., 2003. Contribution of stem cells and differentiated cells to epidermal
tumours. Nat. Rev. Cancer 3, 444-451.
Ozasa, K., Shimizu, Y., Suyama, A., et al., 2012. Studies of the mortality of atomic bomb survivors,
Report 14, 1950-2003: an overview of cancer and noncancer diseases. Radiat. Res. 177, 229243.
Pack, R.J., Al-Ugaily, L.H., Morris, G., 1981. The cells of the tracheobronchial epithelium of themouse: a quantitative light and electron microscope study. J. Anat. 132, 71-84.
Paez, J.G., Janne, P.A., Lee, J.C., et al., 2004. EGFR mutations in lung cancer: correlation withclinical response to gefitinib therapy. Science 304, 1497-1500.
Pan, G.J., Chang, Z.Y., Scholer, H.R., et al., 2002. Stem cell pluripotency and transcription factor
Oct4. Cell Res. 12, 321-329.
Parfitt, A.M., 1983. The physiological and clinical significance of bone histomorphometric data, Bone
histomorphometry: techniques and interpretation. CRC Press Inc., Boca Raton, Florida, pp.
143-223.
Parfitt, A.M., 1988. The composition, structure and remodeling of bone: a basis for the interpretationof bone mineral measurements, Bone mineral measurements by photon absorptiometry:
methodological problems. Leuven University Press, Leuven, Belgium, pp. 9-28.
Parfitt, A.M., 1990. Bone-forming cells in clinical conditions. In: Hall, I. (Ed.), Bone. Telford Press
Inc., N.J., USA, p. 351.
Parise, C.A., Bauer, K.R., Caggiano, V., 2010. Variation in breast cancer subtypes with age andrace/ethnicity. Crit. Rev. Oncol. Hematol. 76, 44-52.
Park, H.S., Goodlad, R.A., Wright, N.A., 1995. Crypt fission in the small intestine and colon. A
mechanism for the emergence of G6PD locus-mutated crypts after treatment with mutagens.
Am. J. Pathol. 147, 1416-1427.
Park, Y., Gerson, S.L., 2005. DNA repair defects in stem cell function and aging. Rev. Med. 56, 495508.
Parkin, D.M., Bray, F., Ferlay J, et al., 2002. Global cancer statistics. CA Cancer J. Clin. 55, 74-108
Parmar, K., Mauch, P., Vergilio, J.A., et al., 2007. Distribution of hematopoietic stem cells in thebone marrow according to regional hypoxia. Proc. Natl. Acad. Sci. U S A. 104, 5431-5436.
Paus, R., Cotsarelis, G., 1999. The biology of hair follicles. N. Engl. J. Med. 341, 491-497.
Pawel, D., Preston, D., Pierce, D., et al., 2008. Improved estimates of cancer site-specific risks for abomb survivors. Radiat. Res. 169, 87-98.
Pazzaglia, S., Mancuso, M., Tanori, M., et al., 2004. Modulation of patched-associated susceptibility
to radiation induced tumorigenesis by genetic background. Cancer Res. 64, 3798-3806.

260

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11170	
  11171	
  11172	
  11173	
  11174	
  11175	
  11176	
  11177	  11178	  11179	
  11180	  11181	
  11182	  11183	  11184	  11185	
  11186	  11187	  11188	  
11189	
  11190	
  
11191	
  
11192	
  
11193	
  
11194	
  
11195	  
11196	
  
11197	  
11198	
  11199	
  11200	
  11201	
  
11202	
  
11203	
  
11204	
  
11205	
  
11206	
  
11207	  
11208	  
11209	
  11210	
  11211	
  11212	
  11213	
  11214	
  11215	
  
11216	  11217	  11218	  11219	
  11220	
  11221	
  Pechoux, C., Gudjonsson, T., Ronnov-Jessen, L., et al., 1999. Human mammary luminal epithelial
cells contain progenitors to myoepithelial cells. Dev. Biol. 206, 88-99.
Penneys, N.S., Fulton, J.E., Jr., Weinstein, G.D., et al., 1970. Location of proliferating cells in humanepidermis. Arch. Dermatol. 101, 323-327.
Pereira, R.F., Halford, K.W., O'Hara, M.D., et al., 1995. Cultured adherent cells from marrow canserve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc. Natl.
Acad. Sci. U.S.A. 92, 4857-4861.
Perez-Losada, J., Balmain, A., 2003. Stem-cell hierarchy in skin cancer. Nat. Rev. Cancer 3, 434-443.
Pesce, M., Scholer, H.R., 2001. Oct-4: gatekeeper in the beginnings of mammalian development.
Stem Cells 19, 271-278.
Pham, D., Kris, M.G., Riely, G.J., et al., 2006. Use of cigarette-smoking history to estimate thelikelihood of mutations in epidermal growth factor receptor gene exons 19 and 21 in lungadenocarcinomas. J. Clin. Oncol. 24, 1700-1704.
Pierce, D.A., Sharp, G.B., Mabuchi, K., 2003. Joint effects of radiation and smoking on lung cancer
risk among atomic bomb survivors. Radiat. Res. 159, 511-520.
Pierce, D.A., Shimizu, Y., Preston, D.L., et al., 1996. Studies of the mortality of atomic bomb
survivors. Report 12, Part I. Cancer: 1950-1990. Radiat. Res. 146, 1-27.
Pietras, E.M., Warr, M.R., Passegue, E., 2011. Cell cycle regulation in hematopoietic stem cells. J.
Cell. Biol. 195, 709-720.
Pignolo, R.J., Kassem, M., 2011. Circulating osteogenic cells: implications for injury, repair, andregeneration. J. Bone Miner. Res. 26, 1685-1693.
Pittenger, M.F., Mackay, A.M., Beck, S.C., et al., 1999. Multilineage potential of adult humanmesenchymal stem cells. Science 284, 143-147.
Plachov, D., Chowdhury, K., Walther, C., et al., 1990. Pax8, a murine paired box gene expressed in
the developing excretory system and thyroid gland. Development 110, 643-651.
Ploemacher, R.E., van Os, R., van Beurden, C.A., et al., 1992. Murine haemopoietic stem cells withlong-term engraftment and marrow repopulating ability are more resistant to gamma-radiation
than are spleen colony forming cells. Int. J. Radiat. Biol. 61, 489-499.
Plumb, M., Harper, K., MacDonald, D., et al., 1997. Ongoing Y-chromosome instability defines subclonal variants in radiation-induced leukaemias in the mouse. Int. J. Radiat. Biol. 72, 1-9.
Pohl, F., Hassel, S., Nohe, A., et al., 2003. Radiation-induced suppression of the Bmp2 signaltransduction pathway in the pluripotent mesenchymal cell line C2C12: an in vitro model for
prevention of heterotopic ossification by radiotherapy. Radiat. Res. 159, 345-350.
Politi, K., Zakowski, M.F., Fan, P.D., et al., 2006. Lung adenocarcinomas induced in mice by mutant
EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to downregulation of the receptors. Genes Dev. 20, 1496-1510.
Poncin, S., Colin, I.M., Gérard, A.C., 2009. Minimal oxidative load: prerequisite for thyroid cellfunction. J. Endocrinol. 201, 161-167.
Postiglione, M.P., Parlato, R., Rodriguez-Mallon, A., et al., 2002. Role of the thyroid-stimulating
hormone receptor signaling in development and differentiation of the thyroid gland. Proc. Natl.
Acad. Sci. U.S.A. 99, 15462-15467.
Potten, C.S., 1975. Epidermal cell production rates. J. Invest. Dermatol. 65, 488-500.
Potten, C.S., 1977. Extreme sensitivity of some intestinal crypt cells to X and gamma irradiation.
Nature 269, 518-521.
Potten, C.S., 1986. The cellular basis of skin injury after cytotoxic insult. Br. J. Cancer Suppl. 7, 4758.
Potten, C.S., 1995. Structure, function and proliferative organisation of mammalian gut. Elsevier,
Amsterdam.
Potten, C.S., 2004a. Keratinocyte stem cells, label-retaining cells and possible genome protection
mechanisms. J. Investig. Dermatol. Symp. Proc. 9, 183-195.
Potten, C.S., 2004b. Radiation, the ideal cytotoxic agent for studying the cell biology of tissues suchas the small intestine. Radiat. Res. 161, 123-136.

261

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11222	
  11223	
  11224	
  11225	
  
11226	
  11227	  11228	  11229	
  11230	
  11231	
  11232	
  11233	
  11234	
  11235	
  
11236	
  11237	  11238	  11239	
  11240	
  11241	
  11242	
  11243	  11244	  11245	  
11246	  11247	  11248	  11249	
  11250	
  11251	
  11252	
  11253	
  11254	  11255	
  
11256	
  11257	  11258	  11259	
  11260	
  11261	
  
11262	
  
11263	  
11264	  
11265	
  11266	  
11267	  
11268	  
11269	
  11270	  11271	
  11272	
  
11273	
  Potten, C.S., Allen, T.D., 1975. Control of epidermal proliferative units (EPUs). An hypothesis basedon the arrangement of neighbouring differentiated cells. Differentiation; research in biologicaldiversity 3, 161-165.
Potten, C.S., Booth, C., 2002. Keratinocyte stem cells: a commentary. J. Invest. Dermatol. 119, 888899.
Potten, C.S., Booth, C., Tudor, G.L., et al., 2003. Identification of a putative intestinal stem cell andearly lineage marker; musashi-1. Differentiation; research in biological diversity 71, 28-41.
Potten, C.S., Gandara, R., Mahida, Y.R., et al., 2009. The stem cells of small intestinal crypts: where
are they? Cell Prolif. 42, 731-750.
Potten, C.S., Grant, H.K., 1998. The relationship between ionizing radiation-induced apoptosis andstem cells in the small and large intestine. Br. J. Cancer 78, 993-1003.
Potten, C.S., Hendry, J.H., 1973. Letter: Clonogenic cells and stem cells in epidermis. Int. J. Radiat.
Biol. Relat. Stud. Phys. Chem. Med. 24, 537-540.
Potten, C.S., Hume, W.J., Reid, P.C., et al., 1978. The segregation of DNA in epithelial stem cells.
Cell 15, 899-906.
Potten, C.S., Li, Y.Q., O'Connor, P.J., et al., 1992. A possible explanation for the differential cancer
incidence in the intestine, based on distribution of the cytotoxic effects of carcinogens in themurine large bowel. Carcinogenesis 13, 2305-2312.
Potten, C.S., Loeffler, M., 1990. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties.
Lessons for and from the crypt. Development 110, 1001-1020.
Potten, C.S., Martin, K., Kirkwood, T.B., 2001. Ageing of murine small intestinal stem cells. Novartis
Foundation symposium 235, 66-79; discussion 79-84, 101-104.
Potten, C.S., Owen, G., Booth, D., 2002. Intestinal stem cells protect their genome by selective
segregation of template DNA strands. Cell Sci. 115, 2381-2388.
Potten, C.S., Wichmann, H.E., Dobek, K., et al., 1985. Cell kinetic studies in the epidermis of mouse.
III. The percent labelled mitosis (PLM) technique. Cell Tissue Kinet. 18, 59-70.
Potten, C.S., Wilson, J., 2007. Adult epithelial stem cells. In: Principles of Tissue Engineering,
Elsevier, Amsterdam.
Powell, S.M., Zilz, N., Beazer-Barclay, Y., et al., 1992. APC mutations occur early during colorectal
tumorigenesis. Nature 359, 235-237.
Prat, A., Parker, J.S., Karginova, O., et al., 2010. Phenotypic and molecular characterization of theclaudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 12, R68.
Preston, D.L., Cullings, H., Suyama, A., et al., 2008. Solid cancer incidence in atomic bomb survivors
exposed in utero or as young children. J. Natl. Cancer Inst. 100, 428-436.
Preston, D.L., Kusumi, S., Tomonaga, M., et al., 1994. Cancer incidence in atomic bomb survivors.
Part III. Leukemia, lymphoma and multiple myeloma, 1950-1987. Radiat. Res. 137, S68-97.
Preston, D.L., Mattsson, A., Holmberg, E., et al., 2002. Radiation effects on breast cancer risk: apooled analysis of eight cohorts. Radiat. Res. 158, 220-235.
Preston, D.L., Pierce, D.A., Shimizu, Y., et al., 2004. Effect of recent changes in atomic bomb
survivor dosimetry on cancer mortality risk estimates. Radiat. Res. 162, 377-389.
Preston, D.L., Pierce, D.A., Shimizu, Y., et al., 2003a. Dose response and temporal patterns ofradiation-associated solid cancer risks. Health Phys. 85, 43-46.
Preston, D.L., Ron, E., Tokuoka, S., et al., 2007. Solid cancer incidence in atomic bomb survivors:
1958-1998. Radiat. Res. 168, 1-64.
Preston, D.L., Shimizu, Y., Pierce, D.A., et al., 2003b. Studies of mortality of atomic bomb survivors.
Report 13: Solid cancer and noncancer disease mortality: 1950-1997. Radiat. Res. 160, 381-407.
Preston, S.L., Wong, W.M., Chan, A.O., et al., 2003c. Bottom-up histogenesis of colorectal
adenomas: origin in the monocryptal adenoma and initial expansion by crypt fission. Cancer
Res. 63, 3819-3825.
Preto, A., Cameselle-Teijeiro, J., Moldes-Boullosa, J., et al., 2004. Telomerase expression andproliferative activity suggest a stem cell role for thyroid solid cell nests. Mod. Pathol. 17, 819826.

262

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11274	  11275	  
11276	  11277	  
11278	  11279	
  11280	  
11281	  
11282	  
11283	  
11284	  
11285	
  11286	  
11287	  
11288	  
11289	
  11290	  
11291	  
11292	  
11293	  
11294	  
11295	  
11296	  
11297	  
11298	
  11299	
  11300	
  11301	
  11302	
  11303	
  11304	
  11305	  
11306	
  11307	  11308	  11309	
  11310	
  11311	
  11312	
  11313	
  11314	  11315	  11316	  11317	  11318	  11319	
  11320	
  11321	
  11322	
  11323	
  11324	
  11325	
  11326	
  Preto, A., Reis-Filho, J.S., Ricardo, S., et al., 2002. P63 expression in papillary and anaplastic
carcinomas of the thyroid gland: lack of an oncogenetic role in tumorigenesis and progression.
Pathol. Res. Pract. 198, 449-454.
Prince, M.E., Ailles, L.E., 2008. Cancer stem cells in head and neck squamous cell cancer. J. Clin.
Oncol. 26, 2871-2875.
Prince, M.E., Sivanandan, R., Kaczorowski, A., et al., 2007. Identification of a subpopulation of cellswith cancer stem cell properties in head and neck squamous cell carcinoma. Proc. Natl. Acad.
Sci. U S A. 104, 973-978.
Prise, K.M., Belyakov, O.V., Newman, H.C., et al., 2002. Non-targeted effects of radiation: bystander
responses in cell and tissue models. Radiat. Prot. Dosim. 99, 223-226.
Prisyazhiuk, A., Pjatak, O.A., Buzanov, V.A., et al., 1991. Cancer in the Ukraine, post-Chernobyl.
Lancet 338, 1334-1335.
Pritchard, D.M., Print, C., O'Reilly, L., et al., 2000. Bcl-w is an important determinant of damageinduced apoptosis in epithelia of small and large intestine. Oncogene 19, 3955-3959.
Przemeck, S.M., Duckworth, C.A., Pritchard, D.M., 2007. Radiation-induced gastric epithelial
apoptosis occurs in the proliferative zone and is regulated by p53, bak, bax, and bcl-2. Am. J.
Physiol. Gastrointest. Liver Physiol. 292, G620-627.
Purton, L.E., Scadden, D.T., 2007. Limiting factors in murine hematopoietic stem cell assays. Cell
Stem Cell 1, 263-270.
Puskin, J.S., Nelson, N.S., Nelson, C.B., 1992. Bone cancer risk estimates. Health Phy. 63, 579-580.
Quinones-Hinojosa, A., Sanai, N., Gonzalez-Perez, O., et al., 2007. The human brain subventricularzone: stem cells in this niche and its organization. Neurosurgery Clin. 18, 15-20, vii.
Raabe, O.G., Culbertson, M.R., White, R.G., et al., 1995. Lifetime radiation effects in beagles injectedwith 226Ra as young adults. In: van Kaick, G., Karaoglou, A., Kellerer, A.M. (Eds.), Health
Effects of Internally Deposited Radionuclides: Emphasis on Radium and Thorium. World
Scientific, Singapore, pp. 313-318.
Rachidi, W., Harfourche, G., Lemaitre, G., et al., 2007. Sensing radiosensitivity of human epidermal
stem cells. Radiother. Oncol. 83, 267-276.
Radzikowska, E., Glaz, P., Roszkowski, K., 2002. Lung cancer in women: age, smoking, histology,
performance status, stage, initial treatment and survival. Population-based study of 20 561
cases. Ann. Oncol. 13, 1087-1093.
Ramdas, L., Giri, U., Ashorn, C.L., et al., 2009. miRNA expression profiles in head and necksquamous cell carcinoma and adjacent normal tissue. Head Neck 31, 642-654.
Ramirez, R.D., Sheridan, S., Girard, L., et al., 2004. Immortalization of human bronchial epithelial
cells in the absence of viral oncoproteins. Cancer Res. 64, 9027-9034.
Randell, S.H., Comment, C.E., Ramaekers, F.C., et al., 1991. Properties of rat tracheal epithelial cells
separated based on expression of cell surface alpha-galactosyl end groups. Am. J. Respir. Cell
Mol. Biol. 4, 544-554.
Rando, T.A., 2007. The immortal strand hypothesis: segregation and reconstruction. Cell 129, 12391243.
Rangarajan, A., Hong, S.J., Gifford, A., et al., 2004. Species- and cell type-specific requirements forcellular transformation. Cancer Cell 6, 171-183.
Rangarajan, A., Weinberg, R.A., 2003. Opinion: Comparative biology of mouse versus human cells:
modelling human cancer in mice. Nat. Rev. Cancer. 3, 952-959.
Rao, B.S., Tano, K., Takeda, S., et al., 2007. Split dose recovery studies using homologous
recombination deficient gene knockout chicken B lymphocyte cells. J. Radiat. Res. 48, 77-85.
Rasko, I., Georgieva, M., Farkas, G., et al., 1993. New patterns of bulk DNA repair in ultraviolet
irradiated mouse embryo carcinoma cells following differentiation. Somatic Cell Molec. Genet.
19, 245-255.
Rawlins, E.L., Hogan, B.L., 2006. Epithelial stem cells of the lung: privileged few or opportunities for
many? Development 133, 2455-2465.
Rawlins, E.L., Ostrowski, L.E., Randell, S.H., et al., 2007. Lung development and repair: contributionof the ciliated lineage. Proc. Natl. Acad. Sci. U S A. 104, 410-417.

263

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11327	  11328	  11329	
  11330	  11331	
  11332	
  11333	
  11334	
  11335	
  11336	
  11337	  11338	  11339	
  11340	
  11341	
  11342	
  11343	
  11344	  11345	  11346	  11347	  11348	  11349	
  11350	
  11351	
  11352	
  11353	
  11354	  11355	  11356	  11357	  11358	  11359	
  11360	
  11361	  11362	  11363	  11364	  11365	
  11366	  11367	  11368	  11369	
  11370	  11371	
  11372	
  11373	  11374	  11375	  11376	  11377	
  Redvers, R.P., Li, A., Kaur, P., 2006. Side population in adult murine epidermis exhibits phentypicand functional characteristics of keratinocyte stem cells. Proc. Natl. Acad. Sci. U S A 103,
12168-12173.
Reis-Filho, J.S., Preto, A., Soares, P., et al., 2003. p63 expression in solid cell nests of the thyroid:
further evidence for a stem cell origin. Mod. Pathol. 16, 43-48.
Renehan, A.G., O'Dwyer, S.T., Haboubi, N.J., et al., 2002. Early cellular events in colorectal
carcinogenesis. Colorectal Dis. 4, 76-89.
Reya, T., Clevers, H., 2005. Wnt signalling in stem cells and cancer. Nature 434, 843-850.
Reya, T., Duncan, A.W., Ailles, L., et al., 2003. A role for Wnt signalling in self-renewal of
haematopoietic stem cells. Nature 423, 409-414.
Reya, T., Morrison, S.J., Clarke, M.F., et al., 2001. Stem cells, cancer, and cancer stem cells. Nature
414, 105-111.
Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from isolated cells of the adultmammalian central nervous system. Science 255, 1707-1710.
Reynolds, S.D., Hong, K.U., Giangreco, A., et al., 2000. Conditional clara cell ablation reveals a selfrenewing progenitor function of pulmonary neuroendocrine cells. Am. J. Physiol. Lung Cell
Mol. Physiol. 278, L1256-1263.
Ricci-Vitiani, L., Lombardi, D.G., Pilozzi, E., et al., 2007. Identification and expansion of humancolon-cancer-initiating cells. Nature 445, 111-115.
Richardson, D., Sugiyama, H., Nishi, N., et al., 2009. Ionizing radiation and leukemia mortality
among Japanese atomic bomb survivors, 1950-2000. Radiat. Res. 172, 368-382.
Richardson, D.B., Cole, S.R., 2012. Model averaging in the analysis of leukemia mortality among
Japanese A-bomb survivors. Radiat. Environ. Biophys. 51, 93-95.
Rieger, K., Marinets, O., Fietz, T., et al., 2005. Mesenchymal stem cells remain of host origin even along time after allogeneic peripheral blood stem cell or bone marrow transplantation. Exp
Hematol. 33, 605-611.
Ries, L.A.G., Smith, M.A., Gurney, L.G., et al., 1999. Cancer incidence and survival among children
and adolescents: United States SEER program 1975-1995. In: Institute, N.C. (Ed.), SEER
Program, 99-4649. National Cancer Institute, Bethesda, MD.
Ris-Stalpers, C., 2006. Physiology and pathophysiology of the DUOXes. Antioxid. Redox. Signal. 8,1563-1572.
Rittie, L., Stoll, S.W., Kang, S., et al., 2009. Hedgehog signaling maintains hair follicle stem cell
phenotype in young and aged human skin. Aging Cell 8, 738-751.
Roberts, S.A., Hendry, J.H., Potten, C.S., 2003. Intestinal crypt clonogens: a new interpretation ofradiation survival curve shape and clonogenic cell number. Cell Prolif. 36, 215-231.
Rocco, J.W., Leong, C.O., Kuperwasser, N., et al., 2006. p63 mediates survival in squamous cell
carcinoma by suppression of p73-dependent apoptosis. Cancer Cell 9, 45-56.
Rock, J.R., Onaitis, M.W., Rawlins, E.L., et al., 2009. Basal cells as stem cells of the mouse trachea
and human airway epithelium. Proc. Natl. Acad. Sci. U S A. 106, 12771-12775.
Ron, E., Lubin, J.H., Shore, R.E., et al., 2012. Thyroid cancer after exposure to external radiation: apooled analysis of seven studies. 1995. Radiat. Res. 178, AV43-60.
Ron, E., Modan, B., Preston, D., et al., 1991. Radiation-induced skin carcinomas of the head and neck.
Radiat. Res. 125, 318-325.
Ron, E., Preston, D.L., Kishikawa, M., et al., 1998. Skin tumor risk among atomic-bomb survivors inJapan. Cancer Causes Control 9, 393-401.
Ron, E., Preston, D.L., Mabuchi, K., et al., 1994. Cancer incidence in atomic bomb survivors. Part IV:
Comparison of cancer incidence and mortality. Radiat. Res. 137, S98-112.
Roscoe, R.J., Deddens, J.A., Salvan, A., et al., 1995. Mortality among Navajo uranium miners. Am. J.
Public Health 85, 535-540.
Roscoe, R.J., Steenland, K., Halperin, W.E., et al., 1989. Lung cancer mortality among nonsmoking
uranium miners exposed to radon daughters. JAMA 262, 629-633.

264

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11378	  11379	
  11380	  11381	  11382	  11383	  11384	  11385	
  11386	  11387	  11388	  11389	
  11390	  
11391	  
11392	  
11393	  
11394	  
11395	  
11396	  11397	  
11398	
  11399	
  11400	
  11401	
  
11402	
  
11403	
  
11404	  
11405	  
11406	  
11407	  
11408	  
11409	
  11410	  11411	
  11412	
  11413	  
11414	  
11415	  
11416	  
11417	  
11418	  
11419	
  11420	
  11421	
  
11422	
  
11423	  
11424	  
11425	  
11426	
  11427	  
11428	
  Rosen, C.J., Ackert-Bicknell, C., Rodriguez, J.P., et al., 2009. Marrow fat and the bone
microenvironment: developmental, functional, and pathological implications. Crit. Rev.
Eukaryot. Gene Expr. 19, 109-124.
Roskams, T., 2006. Liver stem cells and their implication in hepatocellular and cholangiocarcinoma.
Oncogene 25, 3818-3822.
Ross, S.E., Hemati, N., Longo, K.A., et al., 2000. Inhibition of adipogenesis by wnt signaling. Science
289, 950-953.
Rossant, J., Papaioannou, V.E., 1984. The relationship between embryonic, embryonal carcinoma andembryo-derived stem cells. Cell Differentiation 15, 155-161.
Rossi, D.J., Bryder, D., Seita, J., et al., 2007. Deficiencies in DNA damage repair limit the function of
haematopoietic stem cells with age. Nature 447, 725-729.
Rowland R.E., Stehney, A.F,, Lucas, H.F., 1978. Dose-response relationships for female radium dial
workers. Radiat Res. 76, 368-383.
Rowland, R.E., 1997. Bone sarcoma in humans induced by radium: A threshold response?
Radioprotection 32, 331-338.
Rowlands, G.J., Mulatu, W., Nagda, S.M., et al., 1995. Variations in packed red cell volume andtrypanosome prevalence and relationships with reproductive traits in east African Zebu cows
exposed to drug-resistant trypanosomes. Acta Tropica 59, 105-116.
Ruano-Ravina, A., Faraldo-Valles, M.J., Barros-Dios, J.M., 2009. Is there a specific mutation of p53
gene due to radon exposure? A systematic review. Int. J. Radiat. Biol. 85, 614-621.
Rudolph, K.L., Chang, S., Lee, H.W., et al., 1999. Longevity, stress response, and cancer in aging
telomerase-deficient mice. Cell 96, 701-712.
Rundo, J., Keane, A.T., Lucas, H.F., et al., 1986. Current status of the study of 226Ra and 228Ra in
humans at the Center for Human Biology. Strahlentherapie 80, 14-21.
Russell, L.B., Russell, W.L., 1954. An analysis of the changing radiation response of the developing
mouse embryo. J. Cell. Physiol. 43, 103-149.
Russell, W.L., Kelly, E.M., 1982. Mutation frequencies in male mice and the estimation of genetic
hazards of radiation in men. Proc. Natl. Acad. Sci. U S A. 79, 542-544.
Russo, I.H., Russo, J., 1998. Role of hormones in mammary cancer initiation and progression. J.
Mammary Gland Biol. Neoplasia 3, 49-61.
Russo, J., Ao, X., Grill, C., et al., 1999. Pattern of distribution of cells positive for estrogen receptoralpha and progesterone receptor in relation to proliferating cells in the mammary gland. Breast
Cancer Res. Treat 53, 217-227.
Saad, A.G., Kumar, S., Ron, E., et al., 2006. Proliferation activity of human thyroid cells in various
age groups and its correlation with the risk of thyroid cancer after radiation exposure. J. Clin.
Endocrinol. Metab. 91, 2672-2677.
Sadamori, N., Mine, M., Hori, M., 1989. Skin cancer among atom bomb survivors. Lancet 1, 1267.
Sado, T., Kamisaku, H., Kubo, E., 1991. Bone marrow-thymus interactions during thymic
lymphomagenesis induced by fractionated radiation exposure in B10 mice: analysis using bonemarrow transplantation between Thy 1 congenic mice. J. Radiat. Res. 32, 168-180.
Saji, S., Jensen, E.V., Nilsson, S., et al., 2000. Estrogen receptors alpha and beta in the rodent
mammary gland. Proc. Natl. Acad. Sci. U S A. 97, 337-342.
Sakakura, T., Nishizuka, Y., Dawe, C.J., 1976. Mesenchyme-dependent morphogenesis andepithelium-specific cytodifferentiation in mouse mammary gland. Science 194, 1439-1441.
Sakakura, T., Sakagami, Y., Nishizuka, Y., 1979. Persistence of responsiveness of adult mouse
mammary gland to induction by embryonic mesenchyme. Dev. Biol. 72, 201-210.
Sakakura, T., Sakagami, Y., Nishizuka, Y., 1981. Accelerated mammary cancer development by fetal
salivary mesenchyme isografted to adult mouse mammary epithelium. J. Natl. Cancer Inst. 66,
953-959.
Sakakura, T., Sakagami, Y., Nishizuka, Y., 1982. Dual origin of mesenchymal tissues participating inmouse mammary gland embryogenesis. Dev. Biol. 91, 202-207.

265

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11429	
  11430	  11431	
  
11432	
  
11433	  
11434	  
11435	  
11436	  
11437	  
11438	  
11439	
  11440	  
11441	
  
11442	  
11443	  
11444	  
11445	  
11446	  
11447	  
11448	  
11449	
  11450	
  11451	
  
11452	  
11453	  
11454	  
11455	  
11456	  
11457	  
11458	  
11459	
  11460	
  11461	  
11462	  
11463	  
11464	  
11465	
  
11466	  
11467	  
11468	  
11469	
  11470	  
11471	  
11472	  
11473	  
11474	  
11475	  
11476	  
11477	  
11478	  
11479	
  11480	  
11481	
  Samartzis, D., Nishi, N., Hayashi, M., et al., 2011. Exposure to ionizing radiation and development ofbone sarcoma: new insights based on atomic-bomb survivors of Hiroshima and Nagasaki. J.
Bone and Joint Surgery 93, 1008-1015.
Samet, J.M., Kutvirt, D.M., Waxweiler, R.J., et al., 1984. Uranium mining and lung cancer in Navajo
men. N. Engl. J. Med. 310, 1481-1484.
Sanders, C.L., Lauhala, K.E., McDonald, K.E., et al., 1993. Lifespan studies in rats exposed to239
PuO2 aerosol. Health Phys. 64, 509-521.
Sangiorgi, E., Capecchi, M.R., 2008. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40,
915-920.
Sasaki, S., 1991. Influence of the age of mice at exposure to radiation on life-shortening andcarcinogenesis. J. Radiat. Res. 32 Suppl 2, 73-85.
Satagopan, J.M., Offit, K., Foulkes, W., et al., 2001. The lifetime risks of breast cancer in Ashkenazi
Jewish carriers of BRCA1 and BRCA2 mutations. Cancer Epidemiol., Biomarkers &Prevention 10, 467-473.
Sato, T., van Es, J.H., Snippert, H.J., et al., 2011. Paneth cells constitute the niche for Lgr5 stem cells
in intestinal crypts. Nature 469, 415-418.
Sato, T., Vries, R.G., Snippert, H.J., et al., 2009. Single Lgr5 stem cells build crypt-villus structures invitro without a mesenchymal niche. Nature 14, 262-265.
Sauvageau, G., Iscove, N.N., Humphries, R.K., 2004. In vitro and in vivo expansion of hematopoietic
stem cells. Oncogene 23, 7223-7232.
Sauvageau, G., Thorsteinsdottir, U., Eaves, C.J., et al., 1995. Overexpression of HOXB4 inhematopoietic cells causes the selective expansion of more primitive populations in vitro and invivo. Genes Dev. 9, 1753-1765.
Sawey, M.J., Hood, A.T., Burns, F.J., et al., 1987. Activation of c-myc and c-K-ras oncogenes inprimary rat tumors induced by ionizing radiation. Mol. Cell. Biol. 7, 932-935.
Schajowicz, F., 1993. Histological typing of bone tumors. 2nd edition. Springer-Verlag, Heidelberg.
Schedin, P., 2006. Pregnancy-associated breast cancer and metastasis. Nat. Cancer Rev. 6, 281-291.
Scheding, S., Media, J.E., KuKuruga, M.A., et al., 1996. In situ radiation sensitivity of recombinanthuman granulocyte colony-stimulating factor-recruited murine circulating blood and bonemarrow progenitors (colony-forming unit [CFU]-granulocyte-macrophage and CFUmegakaryocyte): evidence for possible biologic differences between mobilized blood and bonemarrow. Blood 88, 472-478.
Schepers, A.G., Snippert, H.J., Stange, D.E., et al., 2012. Lineage tracing reveals Lgr5+ stem cell
activity in mouse intestinal adenomas. Science 337, 730-735.
Schlenker, R.A., Keane, A.T., Unni, K.K., 1989. Comparison of Radium-induced and natural bonesarcomas by historical type, subject age and site of occurence. Scan. Electron Microsc. 1, 377391.
Schluter, H., Paquet-Fifield, S., Gangatirkar, P., et al., 2011. Functional characterization of quiescentkeratinocyte stem cells and their progeny reveals a hierarchical organization in human skinepidermis. Stem Cells 29, 1256-1268.
Schmidt, G.H., Winton, D.J., Ponder, B.A., 1988. Development of the pattern of cell renewal in thecrypt-villus unit of chimaeric mouse small intestine. Development 103, 785-790.
Schneider, T.E., Barland, C., Alex, A.M., et al., 2003. Measuring stem cell frequency in epidermis: aquantitative in vivo functional assay for long-term repopulating cells. Proc. Natl. Acad. Sci. U
S A. 100, 11412-11417.
Schoch, K.G., Lori, A., Burns, K.A., et al., 2004. A subset of mouse tracheal epithelial basal cells
generates large colonies in vitro. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L631-642.
Schonfeld, S.J., Tsareva, Y.V., Preston, D.L., et al., 2012. Cancer mortality following in utero
exposure among offspring of female Mayak Worker Cohort members. Radiat. Res. 178, 160165.
Schonmeyr, B.H., Wong, A.K., Soares, M., et al., 2008. Ionizing radiation of mesenchymal stem cells
results in diminution of the precursor pool and limits potential for multilineage differentiation.
Plast Reconstr. Surg. 122, 64-76.

266

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11482	  
11483	  
11484	  
11485	
  11486	  
11487	  
11488	  
11489	
  11490	  
11491	  
11492	  
11493	  
11494	  
11495	  
11496	  
11497	  
11498	
  
11499	
  11500	
  11501	
  
11502	
  
11503	
  
11504	
  
11505	
  
11506	
  
11507	
  
11508	  
11509	
  11510	
  11511	
  11512	
  11513	
  
11514	  
11515	
  
11516	  
11517	  
11518	  
11519	
  11520	
  11521	
  
11522	
  
11523	
  
11524	
  
11525	
  
11526	
  11527	  
11528	  
11529	
  11530	
  11531	
  
11532	
  

Schwartz, J.L., Kopecky, K.J., Mathes, R.W., et al., 2009. Basal cell skin cancer after total-body
irradiation and hematopoietic cell transplantation. Radiat. Res. 171, 155-163.
Searle, A.G., 1974 Nature and consequences of induced chromosome damage in mammals. Genetics
78, 173-186.
Seed, T.M., 1991. Hematopoietic cell crisis: an early stage of evolving myeloid leukemia following
radiation exposure. J. Radiat. Res. 32, 118-131.
Seed, T.M., Chubb, G.T., Tolle, D.V., et al., 1982. The ultrastructure of radiation-induced endosteal
myelofibrosis in the dog. Scan. Electron Microsc. 377-391.
Seed, T.M., Kaspar, L.V., Fritz, T.E., et al., 1985. Cellular responses in chronic radiation
leukemogenesis. Carcinog. Compr. Surv. 10, 363-379.
Seed, T.M., Kaspar, L.V., Tolle, D.V., et al., 1987. Chronic radiation leukemogenesis: postnatal
hematopathologic effects resulting from in-utero irradiation. Leuk. Res. 11, 171-179.
Seed, T.M., Kaspar, L.V., Tolle, D.V., et al., 1989. Analyses of critical target cell responses during
preclinical phases of evolving chronic radiation-induced myeloproliferative disease:
exploitation of a unique canine model. Multilevel Health Effects research: from molecule toman. Battelle Press, Columbus/Richmond, USA, pp. 245-254.
Seeman, E., Delmas, P.D., 2006. Bone quality--the material and structural basis of bone strength andfragility. New England J. Med. 354, 2250-2261.
Sekido, Y., Fong, K.M., Minna, J.D., 2003. Molecular genetics of lung cancer. Annu. Rev. Med. 54,
73-87.
Sell, S., 2004. Stem cell origin of cancer and differentiation therapy. Critical reviews inoncology/hematology 51, 1-28.
Shackleton, M., Vaillant, F., Simpson, K.J., et al., 2006. Generation of a functional mammary glandfrom a single stem cell. Nature 439, 84-88.
Shames, D.S., Girard, L., Gao, B., et al., 2006. A genome-wide screen for promoter methylation inlung cancer identifies novel methylation markers for multiple malignancies. PLoS Med. 3, e486.
Sharma, S.V., Bell, D.W., Settleman, J., et al., 2007. Epidermal growth factor receptor mutations inlung cancer. Nat. Rev. Cancer. 7, 169-181.
Sheline, G.E., 1969. Thyroid proliferative potential as a function of age. Cell Tissue Kinetics 2, 123127.
Shellabarger, C.J., Bond, V.P., Aponte, G.E., et al., 1966. Results of fractionation and protraction of
total-body radiation on rat mammary neoplasia. Cancer Res. 26, 509-513.
Sheng, H.Z., Westphal, H., 1999. Early steps in pituitary organogenesis. Trends in Genetics : TIG 15,
236-240.
Shepherd, B.E., Guttorp, P., Lansdorp, P.M., et al., 2004. Estimating human hematopoietic stem cell
kinetics using granulocyte telomere lengths. Exp. Hematol. 32, 1040-1050.
Shepherd, B.E., Kiem, H.P., Lansdorp, P.M., et al., 2007. Hematopoietic stem-cell behavior in
nonhuman primates. Blood 110, 1806-1813.
Shi, S., Gronthos, S., 2003. Perivascular niche of postnatal mesenchymal stem cells in human bonemarrow and dental pulp. J. Bone Miner. Res. 18, 696-704.
Shibata, D., 2008. Stem cells as common ancestors in a colorectal cancer ancestral tree. Current
Opinion in Gastroenterol. 24, 59-63.
Shigematsu, H., Gazdar, A.F., 2006. Somatic mutations of epidermal growth factor receptor signaling
pathway in lung cancers. Int. J. Cancer 118, 257-262.
Shih, I.M., Wang, T.L., Traverso, G., et al., 2001. Top-down morphogenesis of colorectal tumors.
Proc. Natl. Acad. Sci. U S A. 98, 2640-2645.
Shimada, Y., Yasukawa-Barnes, J., Kim, R.Y., et al., 1994. Age and radiation sensitivity of rat
mammary clonogenic cells. Radiat. Res. 137, 118-123.
Shimizu, H., Tominaga, S., Nishimura, M., et al., 1984. Comparison of clinico-epidemiologicalfeatures of lung cancer patients with and without a history of smoking. Jpn. J. Clin. Oncol. 14,
595-600.

267

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11533	
  
11534	
  
11535	
  
11536	
  
11537	  
11538	  
11539	
  11540	
  
11541	
  
11542	
  
11543	
  
11544	
  
11545	
  
11546	
  
11547	  
11548	  
11549	
  11550	
  
11551	
  
11552	
  
11553	
  
11554	  
11555	
  
11556	  
11557	  
11558	  
11559	
  11560	
  11561	
  
11562	
  
11563	
  
11564	  
11565	
  11566	  
11567	  
11568	  
11569	
  11570	  
11571	
  
11572	
  
11573	
  
11574	  
11575	  
11576	  
11577	  
11578	  
11579	
  11580	  
11581	
  
11582	  
11583	  
11584	  
11585	
  Shimizu, T., Usuda, N., Yamanda, T., et al., 1993. Proliferative activity of human thyroid tumors
evaluated by proliferating cell nuclear antigen/cyclin immunohistochemical studies. Cancer 71,
2807-2812.
Shimizu, Y., Kato, H., Schull, W.J., 1990. Studies of the mortality of A-bomb survivors. 9. Mortality,
1950-1985: Part 2. Cancer mortality based on the recently revised doses (DS86). Radiat. Res.
121, 120-141.
Shimura, T., Inoue, M., Taga, M., et al., 2002. p53-dependent S-phase damage checkpoint andpronuclear cross talk in mouse zygotes with X-irradiated sperm. Mol. Cell. Biol 22, 2220-2228.
Shiozawa, Y., Taichman, R.S., 2012. Getting blood from bone: an emerging understanding of the rolethat osteoblasts play in regulating hematopoietic stem cells within their niche. Exp. Hematol. 40,
685-694.
Shipitsin, M., Campbell, L.L., Argani, P., et al., 2007. Molecular definition of breast tumor
heterogeneity. Cancer Cell 11, 259-273.
Shizuru, J.A., Negrin, R.S., Weissman, I.L., 2005. Hematopoietic stem and progenitor cells: clinicaland preclinical regeneration of the hematolymphoid system. Ann. Rev. Med. 56, 509-538.
Shoker, B.S., Jarvis, C., Clarke, R.B., et al., 1999. Estrogen receptor-positive proliferating cells in thenormal and precancerous breast. Am. J. Pathol. 155, 1811-1815.
Shore, R., Harley, N., Pasternack, B., et al., 1990. Skin cancer susceptibility among irradiated patients.
J. Am. Acad. Dermatol. 22, 859-860.
Shore, R.E., 1990. Overview of radiation-induced skin cancer in humans. Int. J. Radiat. Biol. 57, 809827.
Shore, R.E., 1992. Issues and epidemiological evidence regarding radiation-induced thyroid cancer.
Radiat. Res. 131, 98-111.
Shore, R.E., 2001. Radiation-induced skin cancer in humans. Med. Pediatr. Oncol. 36, 549-554.
Shore, R.E., Hildreth, N., Woodard, E., et al., 1986. Breast cancer among women given X-ray therapy
for acute postpartum mastitis. J. Natl. Cancer Inst. 77, 689-696.
Shore, R.E., Moseson, M., Xue, X., et al., 2002. Skin cancer after X-ray treatment for scalp ringworm.
Radiat. Res. 157, 410-418.
Shyamala, G., 1997. Roles of estrogen and progesterone in normal mammary gland development:
Insights from progesterone null receptor mutant mice and in situ localization of receptor.
Trends Endocrinol. Metab. 8, 34-39.
Shyamala, G., Chou, Y.-C., Louie, S.G., et al., 2002. Cellular expression of estrogen and progesterone
receptors in mammary glands: Regulation by hormones, development and aging. J. Steroid
Biochem. Mol. Biol. 1655, 1-12.
Sidransky, D., 1996. Is human patched the gatekeeper of common skin cancers? Nat. Genet. 14, 7-8.
Sierra, E., Sahu, S.K., Chiang, Y.W., St. Clair, W.H., Osborne, J.W., 1985. Response of cultured IEC17 normal rat intestinal epithelial cells to X radiation. Radiat. Res. 102, 213-223.
Signoretti, S., Waltregny, D., Dilks, J., et al., 2000. p63 is a prostate basal cell marker and is requiredfor prostate development. Am. J. Pathol. 157, 1769-1775.
Simmons, J.A., Richards, S.R., 1984. Microdosimetry of alpha-irradiated lung. Health Phys. 46, 607616.
Simmons, P.J., Torok-Storb, B., 1991. Identification of stromal cells precursors in human bonemarrow by a novel monoclonal antibody. Blood 78, 55-62.
Singh, S.K., Clarke, I.D., Terasaki, M., et al., 2003. Identification of a cancer stem cell in human brain
tumors. Cancer Res. 63, 5821-5828.
Sivaraman, L., Conneely, O.M., Medina, D., et al., 2001. p53 is a potential mediator of pregnancy andhormone-induced resistance to mammary carcinogenesis. Proc. Natl. Acad. Sci. U S A. 98,
12379-12384.
Smalley, M.J., Titley, J., Paterson, H., et al., 1999. Differentiation of separated mouse mammary
luminal epithelial and myoepithelial cells cultured on EHS matrix analyzed by indirect
immunofluorescence of cytoskeletal antigens. J. Histochem. Cytochem. 47, 1513-1524.
Smith, G.H., 2005. Label-retaining epithelial cells in mouse mammary gland divide asymmetricallyand retain their template DNA strands. Development 132, 681-687.

268

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11586	  
11587	  
11588	  
11589	
  11590	  
11591	
  
11592	  
11593	  
11594	  
11595	  
11596	  
11597	  
11598	
  11599	
  11600	
  11601	
  
11602	
  
11603	
  
11604	  
11605	
  
11606	  
11607	  
11608	  
11609	
  11610	  11611	
  11612	
  11613	  11614	  11615	  
11616	  11617	  11618	  11619	
  11620	
  11621	
  11622	
  11623	
  11624	  11625	
  
11626	
  11627	  11628	  11629	
  11630	  11631	
  11632	
  11633	
  11634	  11635	
  
11636	  11637	  11638	  Smith, G.H., Chepko, G., 2001. Mammary epithelial stem cells. Microsc. Res. Tech. 52, 190-203.
Smith, G.H., Medina, D., 1988. A morphologically distinct candidate for an epithelial stem cell inmouse mammary gland. J. Cell Sci. 90, 173-183.
Smith, G.H., Strickland, P., Daniel, C.W., 2002. Putative epithelial stem cell loss corresponds withmammary growth senescence. Cell Tissue Res. 310, 313-320.
Snippert, H.J., Haegebarth, A., Kasper, M., et al., 2010. Lgr6 marks stem cells in the hair follicle thatgenerate all cell lineages of the skin. Science 327, 1385-1389.
Sokolnikov, M.E., Gilbert, E.S., Preston, D.L., et al., 2008. Lung, liver and bone cancer mortality inMayak workers. Int. J. Cancer 123, 905-911.
Sommer, L., Rao, M., 2002. Neural stem cells and regulation of cell number. Prog. Neurobiol. 66, 118.
Son, B.R., Marquez-Curtis, L.A., Kucia, M., et al., 2006. Migration of bone marrow and cord blood
mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 andhepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 24,
1254-1264.
Sotiropoulou, P.A., Candi, A., Mascre, G., et al., 2010. Bcl-2 and accelerated DNA repair mediates
resistance of hair follicle bulge stem cells to DNA-damage-induced cell death. Nat. Cell. Biol.
12, 572-582.
Soule, H.D., McGrath, C.M., 1986. A simplified method for passage and long-term growth of humanmammary epithelial cells. In Vitro Cell Dev. Biol. 22, 6-12.
Spalding, K.L., Arner, E., Westermark, P.O., et al., 2008. Dynamics of fat cell turnover in humans.
Nature 453, 783-787.
Spalding, K.L., Bhardwaj, R.D., Buchholz, B.A., et al., 2005. Retrospective birth dating of cells inhumans. Cell 122, 133-143.
Spangrude, G.J., Heimfeld, S., Weissman, I.L., 1988. Purification and characterization of mousehematopoietic stem cells. Science 241, 58-62.
Spencer, A., Granter, N., 1999. Leukemia patient-derived lymphoblastoid cell lines exhibit increased
induction of leukemia-associated transcripts following high-dose irradiation. Exp. Hematol. 27,
1397-1401.
Spencer, H., Rampling, D., Aurora, P., et al., 2005. Transbronchial biopsies provide longitudinalevidence for epithelial chimerism in children following sex mismatched lung transplantation.
Thorax 60, 60-62.
Spiers, F.W., Lucas, H.F., Rundo, J., et al., 1983. Leukaemia incidence in the U.S. dial workers.
Health Phys. 44, 65-72.
Spiess, H., 1995. The Ra-224 study: Past, present and future. In: van Kaick, G., Karaoglou, A.,
Kellerer, A.M. (Eds.), Health Effects of Internally Deposited Radionuclides: Emphasis on
Radium and Thorium. World Scientific, Singapore, pp. 157-163.
Stabile, L.P., Lyker, J.S., Gubish, C.T., et al., 2005. Combined targeting of the estrogen receptor andthe epidermal growth factor receptor in non-small cell lung cancer shows enhanced
antiproliferative effects. Cancer Res. 65, 1459-1470.
Stacey, S.N., Sulem, P., Masson, G., et al., 2009. New common variants affecting susceptibility tobasal cell carcinoma. Nat. Genet. 41, 909-914.
Stebbings, J.H., Lucas, H.F., Stehney, A.F., 1984. Mortality from cancers of major sites in female
radium dial workers. Am. J. Indust. Med. 5, 435-459.
Stegelmeier, B.L., Gillett, N.A., Rebar, A.H., et al., 1991. The molecular progression of plutonium239-induced rat lung carcinogenesis: Ki-ras expression and activation. Mol. Carcinog. 4, 43-51.
Stenn, K.S., Paus, R., 2001. Controls of hair follicle cycling. Physiol. Rev. 81, 449-494.
Stephens, P.J., Fu, B., Yang, F., et al., 2011. Massive genomic rearrangement acquired in a single
catastrophic event during cancer development. Cell, 27-40.
Stingl, J., Eirew, P., Ricketson, I., et al., 2006. Purification and unique properties of mammary
epithelial stem cells. Nature 439, 993-997.
Stockwell, H.G., Goldman, A.L., Lyman, G.H., et al., 1992. Environmental tobacco smoke and lungcancer risk in nonsmoking women. J. Natl. Cancer. Inst. 84, 1417-1422.

269

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11639	
  11640	  11641	
  11642	
  11643	
  11644	  11645	  
11646	  11647	  11648	  11649	
  11650	
  11651	
  11652	  11653	
  11654	  11655	  
11656	  11657	  11658	  11659	
  11660	
  11661	
  
11662	  
11663	  
11664	  
11665	
  
11666	  
11667	  
11668	  
11669	
  11670	  11671	  11672	  11673	  11674	  11675	  
11676	  11677	  11678	  11679	
  11680	  
11681	  
11682	  
11683	  
11684	  
11685	
  11686	  11687	  
11688	  
11689	
  11690	
  Storer, J.B., Mitchell, T.J., Fry, R.J., 1988. Extrapolation of the relative risk of radiogenic neoplasms
across mouse strains and to man. Radiat. Res. 114, 331-353.
Stripp, B.R., Maxson, K., Mera, R., et al., 1995. Plasticity of airway cell proliferation and gene
expression after acute naphthalene injury. Am. J. Physiol. 269, L791-799.
Stsjazhko, V.A., Tsyb A.F., Tronko, N.D., et al., 1995. Childhood thyroid cancer since the accident at
Chernobyl. Brit. Med. J. 310, 801.
Suarez, B., Lopez-Abente, G., Martinez, C., et al., 2007. Occupation and skin cancer: the results ofthe HELIOS-I multicenter case-control study. BMC Public Health 7, 180.
Subramanian, J., Govindan, R., 2007. Lung cancer in never smokers: a review. J. Clin. Oncol. 25,
561-570.
Suda, T., 2007. Hematopoietic stem cells and their niche. Acad. Sci., 41-53.
Suda, T., Semenza, G.L., 2011. Metabolic regulation of hematopoietic stem cells in the hypoxic niche.
Cell Stem Cell 9, 298-310.
Sugimoto, M., Takahashi, S., Kotoura, Y., et al., 1993. Osteocyte viability after high-dose irradiationin the rabbit. Clin. Orthop. Relat. Res., 247-252.
Sugrue, T., Brown, J.A., Lowndes, N.F., et al., 2013a. Multiple facets of the DNA damage responsecontribute to the radioresistance of mouse mesenchymal stromal cell lines. Stem Cells 31, 137145.
Sugrue, T., Lowndes, N.F., Ceredig, R., 2013b. Mesenchymal stromal cells: radio-resistant membersof the bone marrow. Immunol. and Cell Biol. 91, 5-11.
Sumimoto, H., 2008. Structure, regulation and evolution of Nox-family NADPH oxidases that
produce reactive oxygen species. FEBS 275, 3249-3277.
Sun, S., Schiller, J.H., Gazdar, A.F., 2007. Lung cancer in never smokers--a different disease. Nat.
Rev. Cancer 7, 778-790.
Sun, Y., Li, H., Yang, H., et al., 2006. Mechanisms controlling embryonic stem cell self-renewal anddifferentiation. Crit. Rev. Eukaryot. Gene Expr. 16, 211-231.
Suratt, B.T., Cool, C.D., Serls, A.E., et al., 2003. Human pulmonary chimerism after hematopoietic
stem cell transplantation. Am. J. Respir. Crit. Care Med. 168, 318-322.
Suzuki, K., Mitsutake, N., Saenko, V., et al., 2011. Dedifferentiation of human primary thyrocytes
into multilineage progenitor cells without gene introduction. PLoS One 6, e19354.
Szilvassy, S.J., Humphries, R.K., Lansdorp, P.M., et al., 1990. Quantitative assay for totipotent
reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc. Natl. Acad.
Sci. U.S.A. 87, 8736-8740.
Tai, M.H., Chang, C.C., Kiupel, M., et al., 2005. Oct4 expression in adult human stem cells: evidencein support of the stem cell theory of carcinogenesis. Carcinogenesis 26, 495-502.
Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic andadult fibroblast cultures by defined factors. Cell 126, 663-676.
Takahashi, S., Sugimoto, M., Kotoura, Y., et al., 1994. Long-term changes in the haversian systems
following high-dose irradiation. An ultrastructural and quantitative histomorphological study. J.
Bone Joint Surg. Am. 76, 722-738.
Takai, H., Smogorzewska, A., de Lange, T., 2003. DNA damage foci at dysfunctional telomeres.
Current biology 13, 1549-1556.
Takamizawa, J., Konishi, H., Yanagisawa, K., et al., 2004. Reduced expression of the let-7
microRNAs in human lung cancers in association with shortened postoperative survival. Cancer
Res. 64, 3753-3756.
Takeuchi, T., Tomida, S., Yatabe, Y., et al., 2006. Expression profile-defined classification of lungadenocarcinoma shows close relationship with underlying major genetic changes andclinicopathologic behaviors. J. Clin. Oncol. 24, 1679-1688.
Tam, P.P., Kanai-Azuma, M., Kanai, Y., 2003. Early endoderm development in vertebrates: lineage
differentiation and morphogenetic function. Curr. Opin. Genet. Dev. 13, 393-400.
Tanaka, H., Yanagisawa, K., Shinjo, K., et al., 2007. Lineage-specific dependency of lungadenocarcinomas on the lung development regulator TTF-1. Cancer Res. 67, 6007-6011.

270

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11691	  
11692	  
11693	  
11694	  
11695	  
11696	  
11697	  
11698	
  11699	
  11700	  11701	  
11702	  
11703	  
11704	  
11705	  
11706	  
11707	  
11708	  
11709	
  11710	  11711	  11712	
  11713	  
11714	  
11715	  
11716	  11717	  11718	  11719	
  11720	
  11721	  
11722	  
11723	  
11724	  11725	  
11726	  11727	  11728	  11729	
  11730	  11731	  
11732	  
11733	  
11734	  
11735	  
11736	  11737	  11738	  11739	
  11740	  
11741	  
11742	
  Tang, X., Lu, X., Guo, W., et al., 2010. Different expression of Sox9 and Runx2 betweenchondrosarcoma and dedifferentiated chondrosarcoma cell line. Eur. J. Cancer Prev. 19, 466471.
Tanori, M., Mancuso, M., Pasquali, E., et al., 2008. PARP-1 cooperates with Ptc1 to suppress
medulloblastoma and basal cell carcinoma. Carcinogenesis 29, 1911-1919.
Tatsumi, S., Ishii, K., Amizuka, N., et al., 2007. Targeted ablation of osteocytes induces osteoporosis
with defective mechanotransduction. Cell Metab. 5, 464-475.
Tawn, J.E., Whitehouse, C.A., Paul Daniel, C., et al., 2003. Somatic cell mutations at the glycophorina locus in erythrocytes of radiation workers from the Sellafield nuclear facility. Radiat. Res.
159, 117-122.
Taylor, J.A., Watson, M.A., Devereux, T.R., et al., 1994. p53 mutation hotspot in radon-associatedlung cancer. Lancet 343, 86-87.
Taylor, R.W., Barron, M.J., Borthwick, G.M., et al., 2003. Mitochondrial DNA mutations in humancolonic crypt stem cells. J. Clin. Invest. 112, 1351-1360.
Terunuma, A., Kapoor, V., Yee, C., et al., 2007. Stem cell activity of human side population andalpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay. Stem Cells 25, 664669.
Terzaghi-Howe, M., 1990. Interactions between cell populations influence expression of thetransformed phenotype in irradiated rat tracheal epithelial cells. Radiat. Res. 121, 242-247.
Terzaghi, M., Nettesheim, P., 1979. Dynamics of neoplastic development in carcinogen-exposedtracheal mucosa. Cancer Res. 39, 3004-3010.
Testa, N.G., Lajtha, L.G., 1973. Comparison of the kinetics of colony forming units in spleen (CFU S)
and culture (CFU C). Br. J. Haematol. 24, 367-376.
Thacker, J., Stretch, A., 1983. Recovery from lethal and mutagenic damage during postirradiation
holding and low-dose-rate irradiations of cultured hamster cells. Radiat. Res. 96, 380-392.
Theeuwes, F., Yum, S.I., 1976. Principles of the design and operation of generic osmotic pumps forthe delivery of semisolid or liquid drug formulations. Ann. Biomed. Eng. 4, 343-353.
Thomas, T., Nowka, K., Lan, L., et al., 2006. Expression of endoderm stem cell markers: evidence forthe presence of adult stem cells in human thyroid glands. Thyroid 16, 537-544.
Thomas, D., Friedman, S., Lin, R.Y., 2008. Thyroid stem cells: lessons from normal development andthyroid cancer. Endocrine-related Cancer 15, 51-58.
Thomas, E.D., Bryant, J.I., Buckner, C.D., et al., 1972 Leukemic transformation of engrafted humanmarrow. Transplant Proc. 4, 567-570.
Thomas, G.P., Baker, S.U., Eisman, J.A., et al., 2001. Changing RANKL/OPG mRNA expression indifferentiating murine primary osteoblasts. J. Endocrinol. 170, 451-460.
Thomas, R.K., Baker, A.C., Debiasi, R.M., et al., 2007. High-throughput oncogene mutation profilingin human cancer. Nat. Genet. 39, 347-351.
Thompson, D.E., Mabuchi, K., Ron, E., et al., 1994. Cancer incidence in atomic bomb survivors. Part
II: Solid tumors, 1958-1987. Radiat. Res. 137, S17-67.
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., et al., 1998. Embryonic stem cell lines derived fromhuman blastocysts. Science 282, 1145-1147.
Thraves, P., Salehi, Z., Dritschilo, A., et al., 1990. Neoplastic transformation of immortalized humanepidermal keratinocytes by ionizing radiation. Proc. Natl. Acad. Sci. U.S.A. 87, 1174-1177.
Tiberio, R., Marconi, A., Fila, C., et al., 2002. Keratinocytes enriched for stem cells are protectedfrom anoikis via an integrin signaling pathway in a Bcl-2 dependent manner. FEBS Lett 524,
139-144.
Tierney, L.A., Hahn, F.F., Lechner, J.F., 1996. p53, erbB-2 and K-ras gene alterations are rare in
spontaneous and plutonium-239-induced canine lung neoplasia. Radiat. Res. 145, 181-187.
Till, J.E., McCulloch, E.A., 1961. A direct measurement of the radiation sensitivity of normal mousebone marrow cells. Radiat. Res. 14, 213-222.
Tirino, V., Desiderio, V., Paino, F., et al., 2011. Human primary bone sarcomas contain CD133+
cancer stem cells displaying high tumorigenicity in vivo. FASEB J 25, 2022-2030.

271

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11743	  
11744	  
11745	  
11746	  
11747	  
11748	  
11749	
  11750	  11751	  11752	  
11753	  
11754	  
11755	  
11756	  11757	  11758	  11759	
  11760	
  11761	  
11762	  
11763	  
11764	  
11765	
  
11766	  
11767	  
11768	  
11769	
  11770	  
11771	  
11772	  
11773	  
11774	  
11775	  
11776	  11777	  11778	  11779	
  11780	  
11781	  
11782	  
11783	  
11784	  
11785	
  11786	  
11787	  
11788	  
11789	
  11790	  
11791	  
11792	  
11793	  
11794	
  Tlsty, T.D., 1998. Cell-adhesion-dependent influences on genomic instability and carcinogenesis.
Curr. Opin. Cell Biol. 10, 647-653.
Toda, S., Aoki, S., Suzuki, K., et al., 2003. Thyrocytes, but not C cells, actively undergo growth andfolliculogenesis at the periphery of thyroid tissue fragments in three-dimensional collagen gel
culture. Cell Tissue Res. 312, 281-289.
Toda, S., Koike, N., Sugihara, H., 2001a. Cellular integration of thyrocytes and thyroid
folliculogenesis: a perspective for thyroid tissue regeneration and engineering. Endocr. J. 48,
407-425.
Toda, S., Koike, N., Sugihara, H., 2001b. Thyrocyte integration, and thyroid folliculogenesis andtissue regeneration: perspective for thyroid tissue engineering. Pathol. Int. 51, 403-417.
Toda, S., Tokuda, Y., Koike, N., et al., 2000. Growth factor-expressing mast cells accumulate at the
thyroid tissue-regenerative site of subacute thyroiditis. Thyroid 10, 381-386.
Toh, C.K., Gao, F., Lim, W.T., et al., 2006. Never-smokers with lung cancer: epidemiologic evidenceof a distinct disease entity. J. Clin. Oncol. 24, 2245-2251.
Tokunaga, M., Land, C.E., Aoki, Y., et al., 1993. Proliferative and nonproliferative breast disease inatomic bomb survivors. Results of a histopathologic review of autopsy breast tissue. Cancer 72,
1657-1665.
Tomasek, L., Rogel, A., Tirmarche, M., et al., 2008. Lung cancer in French and Czech uranium
miners: Radon-associated risk at low exposure rates and modifying effects of time since
exposure and age at exposure. Radiat. Res. 169, 125-137.
Tomkinson, A., Reeve, J., Shaw, R.W., et al., 1997. The death of osteocytes via apoptosis
accompanies estrogen withdrawal in human bone. J. Clin. Endocrinol. Metab. 82, 3128-3135.
Tonon, G., Brennan, C., Protopopov, A., et al., 2005. Common and contrasting genomic profiles
among the major human lung cancer subtypes. Cold Spring Harbor Symposia on Quantitative
Biology 70, 11-24.
Tosh, D., Slack, J.M., 2002. How cells change their phenotype. Nat. Rev. Mol. Cell. Biol. 3, 187-194.
Totafurno, J., Bjerknes, M., Cheng, H., 1987. The crypt cycle. Crypt and villus production in the adultintestinal epithelium. Biophys. J. 52, 279-294.
Toyooka, S., Suzuki, M., Tsuda, T., et al., 2004. Dose effect of smoking on aberrant methylation in
non-small cell lung cancers. Int. J. Cancer 110, 462-464.
Toyooka, S., Toyooka, K.O., Maruyama, R., et al., 2001. DNA methylation profiles of lung tumors.
Mol. Cancer Ther. 1, 61-67.
Toyooka, S., Tsuda, T., Gazdar, A.F., 2003. The TP53 gene, tobacco exposure, and lung cancer. Hum.
Mutat. 21, 229-239.
Travis, L.B., Hill, D.A., Dores, G.M., et al., 2003. Breast cancer following radiotherapy andchemotherapy among young women with Hodgkin disease. JAMA 290, 465-475.
Trempus, C.S., Morris, R.J., Bortner, C.D., et al., 2003. Enrichment for living murine keratinocytes
from the hair follicle bulge with the cell surface marker CD34. J. Invest. Dermatol. 120, 501511.
Trentin, J.J., 1989. Hematopoietic microenvironments: Historical perspective, status, and projections.
Humana Press, Clifton, New Jersey.
Trichopoulos, D., 1990. Hypothesis: does breast cancer originate in utero? Lancet 335, 939-940.
Triffitt, J.T., 1980. The original matrix of bone tissue. J.B. Lippincott Co., Philadelphia.
Trueba, S.S., Auge, J., Mattei, G., et al., 2005. PAX8, TITF1, and FOXE1 gene expression patterns
during human development: new insights into human thyroid development and thyroid
dysgenesis-associated malformations. J. Clin. Endocrinol. Metab. 90, 455-462.
Tsai, Y.C., Lu, Y., Nichols, P.W., et al., 1996. Contiguous patches of normal human mammary
epithelium derived from a single stem cell: Implications for breast carcinogenesis. Cancer Res.
56, 402-404.
Tsukamoto, T., Nishikawa, Y., Yamamoto, M., et al., 1999. Development and distribution of 2amino-1methyl-6phenylimidazo[4,5-bypridine(PhIP)-induced aberrant crypt foci in the rat large
intestine. Jpn. J. Cancer Res., 720-725.

272

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11795	  
11796	  
11797	  
11798	
  11799	
  11800	
  11801	
  
11802	
  
11803	
  
11804	
  
11805	
  
11806	
  
11807	
  
11808	  
11809	
  11810	  11811	
  11812	
  
11813	
  
11814	  
11815	
  
11816	  
11817	  
11818	  
11819	
  11820	
  11821	
  
11822	
  
11823	
  
11824	
  
11825	
  
11826	
  11827	  
11828	  
11829	
  11830	  11831	
  
11832	
  
11833	
  
11834	
  
11835	
  
11836	  
11837	  
11838	  
11839	
  11840	  
11841	
  
11842	
  
11843	
  
11844	  
11845	
  
11846	
  Tsutsumi, S., Shimazu, A., Miyazaki, K., et al., 2001. Retention of multilineage differentiation
potential of mesenchymal cells during proliferation in response to FGF. Biochem. Biophys. Res.
Comm. 288, 413-419.
Tsyb, A.F., Parshkov, E.M., Ivanov, V.K., et al., 1994. Disease incidence of thyroid and the dose
dependence in children and adolescents affected as a result of the Chernobyl accident. Elsevier
Science, New York.
Tuan, R.S., 2003. Cellular signaling in development chondrogenesis: N-cadherin, Wnts, and BMP-2. J.
Bone Joint. Surg. Am. 85, 137-141.
Tucker, S.L., Thames, H.D., Brown, B.W., et al., 1991. Direct analyses of in vivo colony survival
after single and fractionated doses of radiation. Int. J. Radiat. Biol. 59, 777-795.
Tucker, S.L., Withers, H.R., Mason, K.A., et al., 1983. A dose-surviving fraction curve for mousecolonic mucosa. Eur. J. Cancer & Clin. Oncol. 19, 433-437.
Turgeon, J.L., Shyamala, G., Waring, D.W., 2001. PR localization and anterior pituitary cell
populations in vitro in ovariectomized wild-type and pr-knockout mice. Endocrinology 142,4479-4485.
Tuttle, R.M., Onstad, L., Lushnikov, E., et al., 2008. RET/PTC activation is not associated withindividual radiation dose estimates in a pilot study of neopalstic thyroid nodules arising in
Russian children and adults exposed to chernobyl fallout. Thyroid 18, 839-846.
Tuynder, M., Godfrine, S., Cornelis, J.J., et al., 1991. Dose-dependent induction of resistance toterminal differentiation in x-irradiated cultures of normal human keratinocytes. Proc. Natl.
Acad. Sci. U S A. 88, 2638-2642.
Ullrich, R.L., 1983. Tumor induction in BALB/c female mice after fission neutron or gamma
irradiation. Radiat. Res. 93, 506-515.
Ullrich, R.L., 1986. The rate of progression of radiation-transformed mammary epithelial cells isenhanced after low-dose-rate neutron irradiation. Radiat. Res. 105, 68-75.
Ullrich, R.L., Jernigan, M.C., Adams, L.M., 1979. Induction of lung tumors in RFM mice after
localized exposures to X rays or neutrons. Radiat. Res. 80, 464-473.
Ullrich, R.L., Jernigan, M.C., Satterfield, L.C., et al., 1987 Radiation carcinogenesis: time-dose
relationships. Radiat. Res. 111, 179-184.
Ullrich, R.L., Jernigan, M.C., Storer, J.B., 1977. Neutron carcinogenesis. Dose and dose-rate effects
in BALB/c mice. Radiat. Res. 72, 487-498.
Ullrich, R.L., Preston, R.J., 1987. Myeloid leukemia in male RFM mice following irradiation withfission spectrum neutrons or gamma rays. Radiat. Res. 109, 165-170.
Ullrich, R.L., Storer, J.B., 1979. Influence of gamma irradiation on the development of neoplastic
disease in mice. III. Dose-rate effects. Radiat. Res. 80, 325-342.
Unni, K.K., 1996. Dahlin's bone tumors. General aspects and data on 11,087 cases. 5th edition.
Lippincott-Raven, Philadelphia, New York.
UNSCEAR, 1993. Influence of dose and dose rate on stochastic effects of radiation, United Nations
Scientific Committee on the Effects of Atomic Radiation, 1993 Report, Annex F. United
Nations, New York.
UNSCEAR, 2000. Sources and effects of ionizing radiation. United Nations Scientific Committee on
the Effects of Atomic Radiation, New York, USA.
UNSCEAR, 2006. Effects of Ionizing Radiation: United Nations Scientific Committee on the Effectsof Atomic Radiation. Report to the General Assembly, with Scientific Annexes C, D, and E.
United Nations Office at Vienna.
UNSCEAR, 2008. Sources and Effects of Ionizing Radiation. United Nations, New York, NY.
UNSCEAR, 2013. Sources and Effects of Ionizing Radiation. Annex B, Effects of radiation exposureof children. United Nations, New York, NY.
Upton, A.C., 1985. Biological basis for assessing carcinogenic risks of low-level radiation. Carcinog.
Compr. Surv. 10, 381-401.
Upton, A.C., Odell, T.T., Jr., Sniffen, E.P., 1960. Influence of age at time of irradiation on inductionof leukemia and ovarian tumors in RF mice. Proc. Soc. Exp. Biol. Med. 104, 769-772.

273

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11847	  
11848	  
11849	
  11850	
  
11851	
  
11852	
  
11853	
  
11854	  
11855	
  
11856	  
11857	  
11858	  
11859	
  
11860	
  11861	
  
11862	
  
11863	
  
11864	  
11865	
  
11866	  
11867	  
11868	  
11869	
  11870	  
11871	
  
11872	  
11873	  
11874	  
11875	  
11876	  
11877	  
11878	  
11879	
  11880	  
11881	
  
11882	  
11883	  
11884	  
11885	
  11886	  
11887	  
11888	  
11889	
  11890	  
11891	
  
11892	  
11893	  
11894	  
11895	  
11896	  
11897	  
11898	
  11899	
  Upton, A.C., Randolph, M.L., Conklin, J.W., et al., 1970. Late effects of fast neutrons and gammarays in mice as influenced by the dose rate of irradiation: induction of neoplasia. Radiat. Res.
41, 467-491.
Utsuyama, M., Hirokawa, K., 2003. Radiation-induced-thymic lymphoma occurs in young, but not in
old mice. Exp. Mol. Pathol. 74, 319-325.
Vacquier, B., Rogel, A., Leuraud, K., et al., 2009. Radon-associated lung cancer risk among Frenchuranium miners: modifying factors of the exposure–risk relationship. Radiat. Environ. Biophys.
48, 1-9.
Vahakangas, K.H., Bennett, W.P., Castren, K., et al., 2001. p53 and K-ras mutations in lung cancers
from former and never-smoking women. Cancer Res. 61, 4350-4356.
Vahakangas, K.H., Samet, J.M., Metcalf, R.A., et al., 1992. Mutations of p53 and ras genes in radonassociated lung cancer from uranium miners. Lancet 339, 576-580.
van Bekkum, D.W., 1976. The appearance of the multipotential hemopoietic stem cell. SpringerVerlag, New York, NY.
van Bekkum, D.W., 1991. Radiation sensitivity of the hemopoietic stem cell. Radiat. Res. 128, S4-8.
van der Pols, J.C., Williams, G.M., Pandeya, N., et al., 2006. Prolonged prevention of squamous cell
carcinoma of the skin by regular sunscreen use. Cancer Epidemiol. Biomarkers Prev. 15, 25462548.
Van Keymeulen, A., Rocha, A.S., Ousset, M., et al., 2011. Distinct stem cells contribute to mammary
gland development and maintenance. Nature 479, 189-193.
van Loon, B., Markkanen, E., Hubschner, U., 2010. Oxygen as a friend and enemy: How to combat
the mutational potential of 8-oxo-guanine. DNA Repair 9, 604-616.
Van Winkle, L.S., Buckpitt, A.R., Nishio, S.J., et al., 1995. Cellular response in naphthalene-induced
Clara cell injury and bronchiolar epithelial repair in mice. Am. J. Physiol. 269, L800-818.
Vaziri, H., Benchimol, S., 1998. Reconstitution of telomerase activity in normal human cells leads toelongation of telomeres and extended replicative life span. Curr. Biol. 8, 279-282.
Vaziri, H., Dragowska, W., Allsopp, R.C., et al., 1994. Evidence for a mitotic clock in humanhematopoietic stem cells: loss of telomeric DNA with age. Proc. Natl. Acad. Sci. U S A. 91,
9857-9860.
Vigneron, A., Vousden, K.H., 2010. p53, ROS and senescence in the control of aging. Aging 2, 471474.
Villeneuve, P.J., Morrison, H.I., Lane, R., 2007. Radon and lung cancer risk: an extension of themortality follow-up of the Newfoundland fluorspar cohort. Health Phys. 92, 157-169.
Vogelstein, B., Fearon, E.R., Hamilton, S.R., et al., 1988. Genetic alterations during colorectal-tumor
development. New England J. Med. 319, 525-532.
Vriesendorp, H.M., van Bekkum, D.W., 1984. Susceptibilty to total body irradiation. In: Broerse, J.J.,
Macvittie, T.J. (Eds.), Response of Different Species to Total Body Irradiation. Martinus
Nijhoff Publishers, Dordrecht, NL, pp. 43-57.
Wagemaker, G., 1995. Heterogeneity of radiation sensitivity of hemopoietic stem cell subsets. StemCells 13, 257-260.
Waghmare, S.K., Bansal, R., Lee, J., et al., 2008. Quantitative proliferation dynamics and randomchromosome segregation of hair follicle stem cells. Embo. J. 27, 1309-1320.
Wagner, E.R., Luther, G., Zhu, G., et al., 2011. Defective osteogenic differentiation in thedevelopment of osteosarcoma. Sarcoma 2011, 325238.
Wakai, K., Inoue, M., Mizoue, T., et al., 2006. Tobacco smoking and lung cancer risk: an evaluation
based on a systematic review of epidemiological evidence among the Japanese population. Jpn.
J. Clin. Oncol. 36, 309-324.
Wakeford, R., 2008. Childhood leukaemia following medical diagnostic exposure to ionizing
radiation in utero or after birth. Radiat. Prot. Dosim. 132, 166-174.
Wakeford, R., 2012. Radiation effects: Modulating factors and risk assessment -- an overview. Annalsof the ICRP 41, 98-107.
Wakeford, R., Little, M.P., 2003. Risk coefficients for childhood cancer after intrauterine irradiation:a review. Int. J. Radiat. Biol. 79, 293-309.

274

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11900	  11901	  
11902	  
11903	  
11904	  
11905	  
11906	  
11907	  
11908	  
11909	
  11910	  11911	  11912	  
11913	  
11914	  
11915	  
11916	  11917	  
11918	  11919	
  11920	  11921	  
11922	  
11923	  
11924	  
11925	  
11926	  11927	  
11928	  
11929	
  11930	  11931	  
11932	  
11933	  
11934	  
11935	  
11936	  
11937	  
11938	  
11939	
  
11940	  
11941	  
11942	  
11943	  
11944	  
11945	  
11946	  
11947	  
11948	  
11949	
  11950	  11951	  
11952	
  Walburg, H.E., Jr., Upton, A.C., Tyndall, R.L., et al., 1965. Preliminary observations on spontaneousand radiation-induced leukemia in germfree mice. Proc. Soc. Exp. Biol. Med. 118, 11-14.
Wang, D., Jang, D.J., 2009. Protein kinase CK2 regulates cytoskeletal reorganization during ionizing
radiation-induced senescence of human mesenchymal stem cells. Cancer Res. 69, 8200-8207.
Wang, G.Y., Mancianti, M.L., Epstein, E.H. Jr., 2011. Basal cell carcinomas arise from hair follicle
stem cells in ptch1(+/-) mice. Cancer Cell 18, 114-124.
Wang, L., Cui, Y., Lord, B.I., et al., 1996. Gamma-ray-induced cell killing and chromosome
abnormalities in the bone marrow of p53-deficient mice. Radiat. Res. 146, 259-266.
Watanabe, H., Hendry, J.H., 1991. Clonogen number and radiosensitivity in rat thyroid follicles. Int. J.
Radiat. Biol. 128, 222-224.
Watanabe, H., Tanner, M.A., Domann, F.E., et al., 1988. Inhibition of carcinoma formation and of
vascular invasion in grafts of radiation-initiated thyroid clonogens by unirradiated thyroid cells.
Carcinogenesis 9, 1329-1335.
Watson, G.E., Lorimore, S.A., Wright, E.G., 1996. Long-term in vivo transmission of alpha-particleinduced chromosomal instability in murine haemopoietic cells. Int. J. Radiat. Biol. 69, 175-182.
Watson, G.E., Pocock, D.A., Papworth, D., et al., 2001. In vivo chromosomal instability andtransmissible aberrations in the progeny of haemopoietic stem cells induced by high- and lowLET radiations. Int. J. Radiat. Biol. 77, 409-417.
Watt, F.M., Hogan, B.L., 2000. Out of Eden: stem cells and their niches. Science 287, 1427-1430.
Watt, F.M., Jensen, K.B., 2009. Epidermal stem cell diversity and quiescence. EMBO Mol. Med. 1,
260-267.
Watt, T.C., Inskip, P.D., Stratton, K., et al., 2012. Radiation-related risk of basal cell carcinoma: areport from the Childhood Cancer Survivor Study. J. Nat. Cancer Inst. 104, 1240-1250.
Wayman, G.A., Walters, M.J., Kolibaba, K., et al., 2000. CaM kinase IV regulates lineage
commitment and survival of erythroid progenitors in a non-cell-autonomous manner. J. Cell
Biol. 151, 811-824.
Weibel, E., 1963. Morphometry of the human lung. Academic Press.
Weibel, E., 1997. Design and morphomtery of the pulmonary gas exchanger. The Lung: ScientificFoundations 2nd edition, 1147-1157.
Weinstein, G.D., Van Scott, E.J., 1965. Autoradiographic analysis of turnover times of normal andpsoriatic epidermis. J. Invest. Dermatol. 45, 257-262.
Weir, B.A., Woo, M.S., Getz, G., et al., 2007. Characterizing the cancer genome in lungadenocarcinoma. Nature 450, 893-898.
Weiss, H.A., Darby, S.C., Fearn, T., et al., 1995. Leukemia mortality after X-ray treatment forankylosing spondylitis. Radiat. Res. 142, 1-11.
Weissman, I.L., 2000. Stem cells: units of development, units of regeneration, and units in evolution.
Cell 100, 157-168.
Welm, B.E., Tepera, S.B., Venezia, T., et al., 2002. Sca-1(pos) cells in the mouse mammary gland
represent an enriched progenitor cell population. Dev. Biol. 245, 42-56.
Whitehead, R.H., Demmler, K., Rockman, S.P., Watson, N.K.. 1999. Clonogenic growth of epithelial
cells from noraml colonic mucosa from both mice and humans. Gastroenterology 117, 858-865.
Whitehead, R.H., Robinson, P.S., Williams, J.A., et al., 2008. Conditionally immortalized colonicepithelial cell line from Ptk6 null mouse that polarizes and differentiates in vitro. J.
Gastroenterol. Hepatol. 23, 1119-1124.
WHO, 2001. IARC monograph on the evaluation of carcinogenc risks to humans, in: Cancer,
I.A.f.R.o. (Ed.). IARC Press, Lyon, France.
Wick, R.R., Nekolla, E.A., Gaubitz, M., et al., 2008. Increased risk of myeloid leukaemia in patientswith ankylosing spondylitis following treatment with radium-224. Rheumatol. (Oxford) 47,
855-859.
Wick, R.R., Nekolla, E.A., Gossner, W., et al., 1999. Late effects in ankylosing spondylitis patientstreated with 224Ra. Radiat. Res. 152, S8-S11.
Wick, R.R., Nekolla, E.A., Gossner, W., 2005. Long term investigation of late effects in ankylosing
spodylitis patients treated with Ra-224. GS- Forschunszentrum, Neuherberg, pp. 75-81.

275

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
11953	  
11954	  
11955	  
11956	  11957	  
11958	  
11959	  11960	
  11961	  
11962	  
11963	  
11964	  
11965	
  
11966	  
11967	  
11968	  
11969	
  11970	  
11971	  
11972	  
11973	  
11974	  
11975	  
11976	  
11977	  
11978	  
11979	
  11980	  
11981	  
11982	  
11983	  
11984	  
11985	
  
11986	  
11987	  
11988	  
11989	
  11990	  
11991	  
11992	  
11993	  
11994	  
11995	  
11996	  
11997	  
11998	
  11999	
  
12000	
  12001	
  
12002	
  
12003	
  
12004	  Wijnhoven, S.W., Hoogervorst, E.M., de Waard, H., et al., 2007. Tissue specific mutagenic andcarcinogenic responses in NER defective mouse models. Mutat. Res. 614, 77-94.
Willey, J.S., Lloyd, S.A., Robbins, M.E., et al., 2008. Early increase in osteoclast number in mice
after whole-body irradiation with 2 Gy x rays. Radiat. Res. 170, 388-392.
Williams, D., 2009. Radiation carcinogenesis: lessons from chernobyl. Oncogene 27, 9-18.
Williams, J.M., Daniel, C.W., 1983. Mammary ductal elongation: differentiation of myoepitheliumand basal lamina during branching morphogenesis. Dev. Biol. 97, 274-290.
Williams, W.J., Beutler, E., Erslev A.J., Lichman, W.A., 1990. Hematology, 4th edition. McGrawHill Inc, New York, NY.
Wistuba, I., Behrens, C., Virmani, A.K., et al., 2000. High resolution chromosome 3p allelotyping of
human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple,
discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Res. 60,
1949-1960.
Wistuba, I., Lam, S., Behrens, C., et al., 1997. Molecular damage in the bronchial epithelium of
current and former smokers. J. Natl. Cancer Inst. 89, 1366-1373.
Withers, H.R., 1967. Recovery and repopulation in vivo by mouse skin epithelial cells during
fractionated irradiation. Radiat. Res. 32, 227-239.
Withers, H.R., Elkind, M.M., 1969. Radiosensitivity and fractionation response of crypt cells ofmouse jejunum. Radiat. Res. 38, 598-613.
Wood, J.W., Tamagaki, H., Neriishi, S., et al., 1969. Thyroid carcinoma in atomic bomb survivors,
Hiroshima and Nagasaki. Am. J. Epidemiol. 89, 4-14.
Woodward, W.A., Chen, M.S., Behbod, F., et al., 2007. WNT/beta-catenin mediates radiation
resistance of mouse mammary progenitor cells. Proc. Natl. Acad. Sci. U S A. 104, 618-623.
Wu, C.T., Chang, Y.L., Shih, J.Y., et al., 2005. The significance of estrogen receptor beta in 301
surgically treated non-small cell lung cancers. J. Thorac. Cardiovasc. Surg. 130, 979-986.
Wynford-Thomas, D., 2000. Replicative senescence: mechanisms and implications for human cancer.
Pathol. Biol. 48, 301-307.
Xing, M., 2005. BRAF mutation in thyroid cancer. Endocrine-related Cancer 12, 245-262.
Xu, X., Rock, J.R., Lu, Y., et al., 2012. Evidence for type II cells as cells of origin of K-Ras–induced
distal lung adenocarcinoma. Proc. Natl. Acad. Sci. U S A. 109, 4910-4915.
Yager, J.D., Liehr, J.G., 1996. Molecular mechanisms of estrogen carcinogenesis. Annu. Rev.
Pharmacol. Toxicol. 36, 203-232.
Yamashita, S., Saenko, V., 2007. Mechanism of disease: molecular genetics of childhood thyroid
cancers. Nat. Clin. Pract. Endocrinol. Metab. 3, 422-429.
Yamashita, Y.M., Mahowald, A.P., Perlin, J.R., et al., 2007. Asymmetric inheritance of mother versus
daughter centrosome in stem cell division. Science 315, 518-521.
Yanaihara, N., Caplen, N., Bowman, E., et al., 2006. Unique microRNA molecular profiles in lungcancer diagnosis and prognosis. Cancer Cell 9, 189-198.
Yang, L., Peng, R., 2010. Unveiling hair follicle stem cells. Stem Cell Rev 6, 658-664.
Yang, A., Schweitzer, R., Sun, D., et al., 1999. p63 is essential for regenerative proliferation in limb,
craniofacial and epithelial development. Nature 398, 714-718.
Yang, Q., Wesch, H., Mueller, K.M., et al., 2000. Analysis of radon-associated squamous cell
carcinomas of the lung for a p53 gene hotspot mutation. Br. J. Cancer 82, 763-766.
Yang Y, T.L., Lee H, Wu J., 2003. Wnt5a and Wnt5b exhibit distinct activities in coordinatingchondrocyte proliferation and differentiation. Development 130, 1003-1015.
Yatabe, Y., Tavare, S., Shibata, D., 2001. Investigating stem cells in human colon by using
methylation patterns. Proc. Natl. Acad. Sci. U S A. 98, 10839-10844.
Yen, T.H., Wright, N.A., 2006. The gastrointestinal tract stem cell niche. Stem Cell Rev 2, 203-212.
Yin, T., Li, L., 2006. The stem cell niches in bone. J. Clin. Invest. 116, 1195-1201.
Yoshida K., Inoue T., Nojima K., et al., 1997. Calorie restriction reduces the incidence of myeloid
leukemia induced by a single whole-body radiation in H/He mice. Proc. Natl. Acad. Sci. U S A.
94, 2615-2619.

276

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
12005	
  
12006	
  12007	
  
12008	
  
12009	
  12010	  12011	  12012	  12013	  
12014	  12015	  
12016	  12017	  12018	  12019	
  12020	
  12021	  
12022	  
12023	  
12024	  
12025	  
12026	  12027	  
12028	  
12029	
  12030	  12031	  
12032	  
12033	  
12034	  
12035	  
12036	  
12037	  
12038	  
12039	
  12040	  
12041	  
12042	  
12043	  
12044	  
12045	  
12046	  
12047	  
12048	  
12049	
  12050	
  12051	  
12052	  12053	  12054	  12055	
  Young, L.J., Medina, D., DeOme, K.B., et al., 1971. The influence of host and tissue age on life spanand growth rate of serially transplanted mouse mammary gland. Exp. Gerontol. 6, 49-56.
Youssef, K.K., Van Keymeulen, A., Lapouge, G., et al., 2010. Identification of the cell lineage at theorigin of basal cell carcinoma. Nat. Cell. Biol. 12, 299-305.
Yu, H., Wessels, A., Chen, J., et al., 2004. Late gestational lung hypoplasia in a mouse model of theSmith-Lemli-Opitz syndrome. BMC developmental biology 4, 1.
Yu, I.T., Chiu, Y.L., Au, J.S., et al., 2006. Dose-response relationship between cooking fumes
exposures and lung cancer among Chinese nonsmoking women. Cancer Res. 66, 4961-4967.
Yuile, C.L., Gibb, F.R., Morrow, P.E., 1970. Dose-related local and systemic effects of inhaledplutonium-238 and plutonium-239 dioxide in dogs. Radiat. Res. 44, 821-834.
Yumoto, K., Globus, R.K., Mojarrab, R., et al., 2010. Short-term effects of whole-body exposure to(56)fe ions in combination with musculoskeletal disuse on bone cells. Radiat. Res. 173, 494504.
Zandstra, P.W., Conneally, E., Petzer, A.L., et al., 1997. Cytokine manipulation of primitive humanhematopoietic cell self-renewal. Proc. Natl. Acad. Sci. U S A. 94, 4698-4703.
Zanetti, R., Rosso, S., Martinez, C., et al., 2006. Comparison of risk patterns in carcinoma andmelanoma of the skin in men: a multi-centre case-case-control study. Br. J. Cancer 94, 743-751.
Zannini, M., Avantaggiato, V., Biffali, E., et al., 1997. TTF-2, a new forkhead protein, shows atemporal expression in the developing thyroid which is consistent with a role in controlling theonset of differentiation. Embo. J. 16, 3185-3197.
Zaragosi, L.G., Ailhaud, G., Dani, C., , 2006. Autocrine FGF2 signalling is critical for self renewal
multipoint adipose-derived stem cells. Stem Cells 24, 2412-2419.
Zarrilli, R., Formisano, S., Di Jeso, B., 1990. Hormonal regulation of thyroid peroxidase in normal
and transformed rat thyroid cells. Mol. Endocrinol. 4, 39-45.
Zavadil, J., Bottinger, E.P., 2005. TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 24,5764-5774.
Zavadil, J., Cermak, L., Soto-Nieves, N., et al., 2004. Integration of TGF-beta/Smad andJagged1/Notch signalling in epithelial-to-mesenchymal transition. Embo. J. 23, 1155-1165.
Zeki, S.S., Graham, T.A., Wright, N.A., 2011. Stem cells and their implications for colorectal cancer.
Gastroenterol. & Hepatol. 8, 90-100.
Zeps, N., Bentel, J.M., Papadimitriou, J.M., et al., 1998. Estrogen receptor-negative epithelial cells inmouse mammary gland development and growth. Differentiation 62, 221-226.
Zeps, N., Dawkins, H.J.S., Papadimitriou, J.M., et al., 1996. Detection of a population of long-lived
cells in mammary epithelium of the mouse. Cell Tissue Res. 286, 525-536.
Zha, S., Alt, F.W., Cheng, H.L., et al., 2007. Defective DNA repair and increased genomic instability
in Cernunnos-XLF-deficient murine ES cells. PNAS 104, 4518-4523.
Zhang, J., Niu, C., Ye, L., et al., 2003. Identification of the haematopoietic stem cell niche and controlof the niche size. Nature 425, 836-841.
Zhao, R., Xi, R., 2010. Stem cell competition for niche occupancy: emerging themes and mechanisms.
Stem Cell Reviews 6, 345-350.
Zheng, D., Limmon, G.V., Yin, L., et al., 2012. Regeneration of alveolar type I and II cells fromscgb1a1-expressing cells following severe pulmonary damage induced by bleomycin andinfluenza. PLoS ONE 7, e48451.
Zhong, L., Goldberg, M.S., Gao, Y.T., et al., 1999. A case-control study of lung cancer andenvironmental tobacco smoke among nonsmoking women living in Shanghai, China. Cancer
Causes Control 10, 607-616.
Zhou, H., Randers-Pehrson, G., Waldren, C.A., et al., 2004. Radiation-induced bystander effect andadaptive response in mammalian cells. Adv. Space Res. 34, 1368-1372.
Zhou, S., Morris, J.J., Barnes, Y., et al., 2002. Bcrp1 gene expression is required for normal numbersof side population stem cells in mice, and confers relative protection to mitoxantrone inhematopoietic cells in vivo. Proc. Natl. Acad. Sci. U S A. 99, 12339-12344.

277

DRAFT REPORT FOR CONSULTATION: DO NOT REFERENCE
12056	  12057	  12058	  12059	
  12060	
  12061	  
12062	  
12063	  
12064	  
12065	
  12066	  
12067	  
12068	  
12069	
  12070	  
12071	  
12072	  
12073	  
12074	
  Zhou, S., Schuetz, J.D., Bunting, K.D., et al., 2001. The ABC transporter Bcrp1/ABCG2 is expressedin a wide variety of stem cells and is a molecular determinant of the side-population phenotype.
Nature Med. 7, 1028-1034.
Zhu, L.L., Wu, L.Y., Yew, D.T., et al., 2005. Effects of hypoxia on the proliferation anddifferentiation of NSCs. Mol. Neurobiol. 31, 231-242.
Ziegler, B.L., Valtieri, M., Porada, G.A., et al., 1999. KDR receptor: a key marker defining
hematopoietic stem cells. Science 285, 1553-1558.
Zochbauer-Muller, S., Fong, K.M., Virmani, A.K., et al., 2001. Aberrant promoter methylation ofmultiple genes in non-small cell lung cancers. Cancer Res. 61, 249-255.
Zochbauer-Muller, S., Gazdar, A.F., Minna, J.D., 2002. Molecular pathogenesis of lung cancer. Ann.
Rev. Physiol. 64, 681-708.
Zochbauer-Muller, S., Lam, S., Toyooka, S., et al., 2003. Aberrant methylation of multiple genes in
the upper aerodigestive tract epithelium of heavy smokers. Int. J. Cancer 107, 612-616.
zur Hausen, H., 2009. Childhood leukemias and other hematopoietic malignancies: interdependence
between an infectious event and chromosomal modifications. Int. J. Cancer 125, 1764-1770.
Zyuzikov, N.A., Coates, P.J., Parry, J.M., et al., 2011. Lack of nontargeted effects in murine bonemarrow after low-dose in vivo X irradiation. Radiat. Res. 175, 322-327.

278